Stabilization Of Perhydrolases

ABSTRACT

Disclosed herein are enzyme powders comprising a spray-dried formulation of at least one CE-7 esterase, at least one oligosaccharide excipient, and optionally at least one surfactant. Also disclosed herein is a process for producing peroxycarboxylic acids from carboxylic acid esters using the aforementioned enzyme powders. Further, disinfectant and laundry care formulations comprising the peracids produced by the processes described herein are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of pending U.S. patent application Ser.No. 12/572,070, filed Oct. 1, 2009, which claims the benefit of U.S.Provisional Application Nos. 61/102,505; 61/102,512; 61/102,514;61/102,520; 61/102,531; and 61/102,539; each filed Oct. 3, 2008, each ofwhich incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates to the field of enzymatic peracid synthesis andin situ enzyme catalysis. At least one peroxycarboxylic acid is producedat sufficient concentrations as to be efficacious for the disinfectionor sanitization of surfaces, medical instrument sterilization, foodprocessing equipment sterilization, and suitable for use in textile andlaundry care applications such as bleaching, destaining, deodorizing,disinfection or sanitization.

BACKGROUND OF THE INVENTION

Peracid compositions have been reported to be effective antimicrobialagents. Methods to clean, disinfect, and/or sanitize hard surfaces, meatproducts, living plant tissues, and medical devices against undesirablemicrobial growth have been described (e.g., U.S. Pat. No. 6,545,047;U.S. Pat. No. 6,183,807; U.S. Pat. No. 6,518,307; U.S. Pat. No.5,683,724; and U.S. Patent Application Publication No. 2003/0026846).Peracids have also been reported to be useful in preparing bleachingcompositions for laundry detergent applications (U.S. Pat. No.3,974,082; U.S. Pat. No. 5,296,161; and U.S. Pat. No. 5,364,554).

Peracids can be prepared by the chemical reaction of a carboxylic acidand hydrogen peroxide (see Organic Peroxides, Daniel Swern, ed., Vol. 1,pp 313-516; Wiley Interscience, New York, 1971). The reaction is usuallycatalyzed by a strong inorganic acid, such as concentrated sulfuricacid. The reaction of hydrogen peroxide with a carboxylic acid is anequilibrium reaction, and the production of peracid is favored by theuse of an excess concentration of peroxide and/or carboxylic acid, or bythe removal of water.

Some peracid-based disinfectants or bleaching agents are comprised of anequilibrium mixture of peracid, hydrogen peroxide, and the correspondingcarboxylic acid. One disadvantage of these commercial peracid cleaningsystems is that the peracid is oftentimes unstable in solution overtime. One way to overcome the stability problem is to generate theperacid prior to use by combining multiple reaction components that areindividually stable for extended periods of time. Preferably, theindividual reaction components are easy to store, relatively safe tohandle, and capable of quickly producing an efficacious concentration ofperacid upon mixing.

The CE-7 family of carbohydrate esterases has recently been reported tohave perhydrolase activity. These “perhydrolase” enzymes have beendemonstrated to be particularly effective for producing peracids from avariety of carboxylic acid ester substrates when combined with a sourceof peroxygen (See WO2007/070609 and U.S. Patent Application PublicationNos. 2008/0176299 and 2008/176783 to DiCosimo et al.; each hereinincorporated by reference in their entireties). Some members of the CE-7family of carbohydrate esterases have been demonstrated to haveperhydrolytic activity sufficient to produce 4000-5000 ppm peraceticacid from acetyl esters of alcohols, diols, and glycerols in 1 minuteand up to 9000 ppm between 5 minutes and 30 minutes once the reactioncomponents were mixed (DiCosimo et al., U.S. Patent ApplicationPublication No. 2009/0005590).

The enzymatic peracid generation system described by U.S. 2009/0005590to DiCosimo et al. is typically based on the use of multiple reactioncomponents that remain separated until the peracid solution is needed.Using this approach overcomes the peracid instability issues associatedwith storage of many peracid-based disinfectants and bleaching agents.However, specific formulations that provide long term stability ofperhydrolase activity when using multicomponent formulations comprisingCE-7 carbohydrate esterases remains to be addressed. Of particularconcern is the long term storage stability of a CE-7 enzyme havingperhydrolysis activity when stored in an organic liquid or solventhaving a log P (i.e., the logarithm of the partition coefficient of asubstance between octanol and water, where P equals[solute]_(octanol)/[solute]_(water)) of less than two. Several of theorganic ester substrates previous described by DiCosimo et al. have logP values of less than two.

Organic liquids or solvents can be deleterious to the activity ofenzymes, either when enzymes are suspended directly in organic liquidsor solvents, or when miscible organic/aqueous single phase liquids orsolvents are employed. Two literature publications that review theeffects of organic solvents on enzyme activity and structure are: (a) C.Laane et al., Biotechnol. Bioeng. 30:81-87 (1987) and (b) Cowan, D. A.and Plant, A., Biocatalysis in Organic Phase Systems., Ch. 7 inBiocatalysis at Extreme Temperatures, Kelly, R. W. W. and Adams, M.,eds., Amer. Chem. Soc. Symposium Series, Oxford University Press, NewYork, N.Y., pp 86-107 (1992). Cowan and Plant, supra, note (on page 87)that the art generally recognizes that there is little or no value inusing organic solvents having a log P≦2 to stabilize intracellularenzymes in an organic phase system. Organic solvents having a log Pbetween two and four can be used on a case-by-case basis dependent onenzyme stability, and those having a log P>4 are generally useful inorganic phase systems.

Cowan and Plant, supra, further note (on page 91) that the effect ofdirect exposure of an enzyme dissolved in a single-phase organic-aqueoussolvent depends on solvent concentration, solvent/enzyme surface groupinteractions, and solvent/enzyme hydration shell interactions. Because asolvent's log P value must be sufficiently low so that the solvent isfully miscible with the aqueous phase to produce a single-phase, asingle-phase organic-aqueous solvent containing a low log P organicsolvent usually has a negative effect on enzyme stability except in loworganic solvent concentration applications. Triacetin is reported tohave a log P of 0.25 (Y. M. Gunning, et al., J. Agric. Food Chem.48:395-399 (2000)), similar to that of ethanol (log P −0.26) andisopropanol (log P 0.15) (Cowan and Plant); therefore the storage ofenzyme powder in triacetin would be expected to result in unacceptableloss of enzyme activity, as would the use of additional cosolvents withlog P<2 (e.g., cyclohexanone, log P=0.94) (Cowan and Plant);1,2-propanediol, log P=−1.41 (Gunning, et al.); 1,3-propanediol, logP=−1.3 (S-J. Kuo, et al., J. Am. Oil Chem. Soc. 73:1427-1433 (1996);diethylene glycol butyl ether, log P=0.56 (N. Funasaki, et al., J. Phys.Chem. 88:5786-5790 (1984); triethyleneglycol, log P=−1.75 (L. Braeken,et al., Chem Phys Chem 6:1606-1612 (2005)).

Thus, the problem to be solved is to formulate a product using a mixtureof a peracid-generating enzyme in an organic ester substrate employedfor peracid production, where the enzyme retains significantperhydrolase activity even when stored in a mixture with the carboxylicacid ester substrate.

SUMMARY OF THE INVENTION

The stated problem has been solved by the discovery of a process forspray-drying an aqueous formulation comprising at least one enzymestructurally classified as a CE-7 enzyme and having perhydrolysisactivity, wherein the formulation further comprises an oligosaccharideexcipient that stabilizes the perhydrolase activity when the spray-driedformulation (an enzyme powder) is combined with an carboxylic acid estersubstrate employed for peracid production.

In one aspect, a process to stabilize the perhydrolysis activity of anenzyme when present in a formulation comprised of said enzyme and acarboxylic acid ester is provided, the process comprising:

-   -   (a) providing an aqueous formulation comprising at least one        enzyme structurally classified as a CE-7 enzyme and having        perhydrolysis activity, at least one oligosaccharide excipient,        and optionally at least one surfactant; and    -   (b) spray-drying the aqueous formulation of (a) to produce an        enzyme powder which substantially retains the perhydrolysis        activity of the at least one enzyme when present in a        formulation comprised of a carboxylic acid ester and the enzyme        powder.

Another aspect is for an enzyme powder comprising a spray-driedformulation of at least one enzyme structurally classified as a CE-7enzyme and having perhydrolysis activity and at least oneoligosaccharide excipient, and optionally at least one surfactant;wherein the enzyme powder substantially retains the perhydrolysisactivity of the at least one enzyme when present in a formulationcomprised of a carboxylic acid ester and the enzyme powder.

A further aspect is for a formulation comprising the enzyme powderdiscussed above mixed with a carboxylic acid ester. In another aspect,the formulation comprises the enzyme powder mixed with a carboxylic acidester selected from the group consisting of monoacetin, diacetin,triacetin, monopropionin, dipropionin, tripropionin, monobutyrin,dibutyrin, tributyrin, and mixtures thereof.

An additional aspect is for a process to produce a disinfectantformulation comprising:

-   -   (a) providing an aqueous formulation comprising at least one        enzyme structurally classified as a CE-7 enzyme and having        perhydrolysis activity, at least one oligosaccharide excipient,        and optionally at least one surfactant;    -   (b) spray-drying the aqueous formulation of (a) to produce an        enzyme powder; and    -   (c) combining the enzyme powder of (b) with a carboxylic acid        ester and an aqueous solution comprising a source of peroxygen.

Another aspect is for a process for producing a peroxycarboxylic acidfrom a carboxylic acid ester comprising

-   -   (a) providing a set of reaction components, said components        comprising:        -   (1) a formulation comprising:            -   i) the enzyme powder discussed above; and            -   ii) a carboxylic acid ester; and        -   (2) a source of peroxygen; and    -   (b) combining said reaction components under suitable aqueous        reaction conditions whereby a peroxycarboxylic acid is produced.

A further aspect is for a process to disinfect or sanitize a hardsurface or inanimate object using an enzymatically-producedperoxycarboxylic acid composition, said process comprising:

-   -   (a) providing a set of reaction components, said components        comprising:        -   (1) a formulation comprising:            -   i) the enzyme powder discussed above; and            -   ii) a carboxylic acid ester; and        -   (2) a source of peroxygen;    -   (b) combining said reaction components under suitable aqueous        reaction conditions whereby a peroxycarboxylic acid product is        formed;    -   (c) optionally diluting said peroxycarboxylic acid product; and    -   (d) contacting said hard surface or inanimate object with the        peroxycarboxylic acid produced in step (b) or step (c) whereby        said surface or said inanimate object is disinfected.

A further aspect is for a process for treating an article of clothing ora textile for bleaching, stain removal, odor reduction, sanitization ordisinfection using an enzymatically-produced peroxycarboxylic acidcomposition, said process comprising:

-   -   (a) providing a set of reaction components, said components        comprising:        -   (1) a formulation comprising            -   i) the enzyme powder discussed above; and            -   ii) a carboxylic acid ester; and        -   (2) a source of peroxygen;    -   (b) combining said reaction components under suitable aqueous        reaction conditions whereby a peroxycarboxylic acid product is        formed;    -   (c) optionally diluting said peroxycarboxylic acid product; and    -   (d) contacting said article of clothing or textile with the        peroxycarboxylic acid produced in step (b) or step (c);        wherein said article of clothing or textile is destained,        deodorized, disinfected, bleached, sanitized or a combination        thereof.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§1.821-1.825(“Requirements for patent applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EuropeanPatent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules5.2 and 49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the deduced amino acid sequence of a cephalosporin Cdeacetylase from Bacillus subtilis ATCC® 31954™.

SEQ ID NO:2 is the deduced amino acid sequence of a cephalosporin Cdeacetylase from B. subtilis ATCC® 6633™.

SEQ ID NO:3 is the deduced amino acid sequence of a cephalosporin Cdeacetylase from B. licheniformis ATCC® 14580™.

SEQ ID NO:4 is the deduced amino acid sequence of an acetyl xylanesterase from B. pumilus PS213.

SEQ ID NO:5 is the deduced amino acid sequence of an acetyl xylanesterase from Clostridium thermocellum ATCC® 27405™.

SEQ ID NO:6 is the deduced amino acid sequence of an acetyl xylanesterase from Thermotoga neapolitana.

SEQ ID NO:7 is the deduced amino acid sequence of an acetyl xylanesterase from Thermotoga maritima MSB8.

SEQ ID NO:8 is the deduced amino acid sequence of an acetyl xylanesterase from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO:9 is the deduced amino acid sequence of a cephalosporin Cdeacetylase from Bacillus sp. NRRL B-14911. It should be noted that thenucleic acid sequence encoding the cephalosporin C deacetylase fromBacillus sp. NRRL B-14911 as reported in GENBANK® Accession numberZP_(—)01168674 appears to encode a 15 amino acid N-terminal additionthat is likely incorrect based on sequence alignments with othercephalosporin C deacetylases and a comparison of the reported length(340 amino acids) versus the observed length of other CAH enzymes(typically 318-325 amino acids in length; see co-owned U.S. Pat. No.8,105,810). As such, the deduced amino acid sequence reported herein forthe cephalosporin C deacetylase sequence from Bacillus sp. NRRL B-14911does not include the N-terminal 15 amino acids as reported underGENBANK® Accession number ZP_(—)01168674.

SEQ ID NO:10 is the deduced amino acid sequence of a cephalosporin Cdeacetylase from Bacillus halodurans C-125.

SEQ ID NO:11 is the deduced amino acid sequence of a cephalosporin Cdeacetylase from Bacillus clausii KSM-K16.

SEQ ID NO:12 is the deduced amino acid sequence of a Bacillus subtilisATCC® 29233™ cephalosporin C deacetylase (CAH).

SEQ ID NO:13 is the deduced amino acid sequence of aThermoanearobacterium saccharolyticum cephalosporin C deacetylase.

SEQ ID NO:14 is the deduced amino acid sequence of a Thermotogalettingae acetyl xylan esterase.

SEQ ID NO:15 is the deduced amino acid sequence of a Thermotogapetrophila acetyl xylan esterase.

SEQ ID NO:16 is the deduced amino acid sequence of a first acetyl xylanesterase from Thermotoga sp. RQ2 described herein as “RQ2(a)”.

SEQ ID NO:17 is the deduced amino acid sequence of a second acetyl xylanesterase from Thermotoga sp. RQ2 described herein as “RQ2(b)”.

SEQ ID NO:18 is the amino acid sequence of the region encompassing aminoacids residues 118 through 299 of SEQ ID NO:1.

SEQ ID NO:19 is the deduced amino acid sequence of a Thermotoganeapolitana acetyl xylan esterase variant from co-owned U.S. Pat. No.8,062,875 (incorporated herein by reference in its entirety), where theXaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO:20 is the deduced amino acid sequence of a Thermotoga maritimaMSB8 acetyl xylan esterase variant from co-owned U.S. Pat. No.8,062,875, where the Xaa residue at position 277 is Ala, Val, Ser, orThr.

SEQ ID NO:21 is the deduced amino acid sequence of a Thermotogalettingae acetyl xylan esterase variant from co-owned U.S. Pat. No.8,062,875, where the Xaa residue at position 277 is Ala, Val, Ser, orThr.

SEQ ID NO:22 is the deduced amino acid sequence of a Thermotogapetrophila acetyl xylan esterase variant from co-owned U.S. Pat. No.8,062,875, where the Xaa residue at position 277 is Ala, Val, Ser, orThr.

SEQ ID NO:23 is the deduced amino acid sequence of a Thermotoga sp. RQ2acetyl xylan esterase variant derived from “RQ2(a)” from co-owned U.S.Pat. No. 8,062,875, where the Xaa residue at position 277 is Ala, Val,Ser, or Thr.

SEQ ID NO:24 is the deduced amino acid sequence of a Thermotoga sp. RQ2acetyl xylan esterase variant derived from “RQ2(b)” from co-owned U.S.Pat. No. 8,062,875, where the Xaa residue at position 278 is Ala, Val,Ser, or Thr.

SEQ ID NO:25 is the deduced amino acid sequence of aThermoanaerobacterium sp. JW/SL YS485 acetyl xylan esterase.

SEQ ID NO:26 is the coding region of a kanamycin resistance gene (kan)from Streptomyces kanamyceticus.

SEQ ID NO:27 is plasmid pKD13, which contains the kanamycin resistancegene.

SEQ ID NO:28 is a forward primer used to clone katG from plasmid pKD13.

SEQ ID NO:29 is a reverse primer used to clone katG from plasmid pKD13.

SEQ ID NO:30 is the PCR product of the katG amplification from plasmidpKD13 using the primers of SEQ ID NO:28 and SEQ ID NO:29.

SEQ ID NO:31 is the coding region of the catalase-peroxidase gene(katG).

SEQ ID NO:32 is the deduced amino acid sequence of katG.

SEQ ID NO:33 is plasmid pKD46, which contains the λ-Red recombinasegenes.

SEQ ID NO:34 is a forward primer used to confirm disruption of katG. SEQID NO:35 is a reverse primer used to confirm disruption of katG.

SEQ ID NO:36 is the temperature-sensitive plasmid pCP20, which containsthe FLP recombinase.

SEQ ID NO:37 is a forward primer used to clone katE from plasmid pKD13.

SEQ ID NO:38 is a reverse primer used to clone katE from plasmid pKD13.

SEQ ID NO:39 is the PCR product of the katE amplification from plasmidpKD13 using the primers of SEQ ID NO:37 and SEQ ID NO:38.

SEQ ID NO:40 is the coding region of the catalase HPII gene (katE).

SEQ ID NO:41 is the deduced amino acid sequence of katE.

SEQ ID NO:42 is a forward primer used to confirm disruption of katE.

SEQ ID NO:43 is a reverse primer used to confirm disruption of katE.

SEQ ID NO:44 is a coding region of a gene encoding acetyl xylan esterasefrom Thermotoga neapolitana as reported in GENBANK® (accession#AE000512).

SEQ ID NO:45 is a forward primer used to amplify the acetyl xylanesterase gene from Thermotoga neapolitana.

SEQ ID NO:46 is a reverse primer used to amplify the acetyl xylanesterase gene from Thermotoga neapolitana.

SEQ ID NO:47 is the PCR product of the acetyl xylan esteraseamplification using the primers of SEQ ID NO:45 and SEQ ID NO:46.

SEQ ID NO:48 is a gene encoding acetyl xylan esterase from Thermotogamaritima MSB8 as reported in GENBANK® (accession #NP_(—)227893.1).

SEQ ID NO:49 is a forward primer used to amplify the acetyl xylanesterase gene from Thermotoga maritima.

SEQ ID NO:50 is a reverse primer used to amplify the acetyl xylanesterase gene from Thermotoga maritima.

SEQ ID NO:51 is the PCR product of the acetyl xylan esteraseamplification using the primers of SEQ ID NO:49 and SEQ ID NO:50.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are enzyme powders comprising a spray-dried formulationof at least one CE-7 carbohydrate esterase having perhydrolysisactivity, at least one oligosaccharide excipient, and optionally atleast one surfactant. Also disclosed herein is a process for producingperoxycarboxylic acids from carboxylic acid esters using theaforementioned enzyme powders. Further, disinfectant formulationscomprising the peracids produced by the processes described herein areprovided.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an” and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof. The term“comprising” is intended to include embodiments encompassed by the terms“consisting essentially of” and “consisting of”. Similarly, the term“consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the terms “substrate”, “suitable substrate”, and“carboxylic acid ester substrate” interchangeably refer specifically to:

-   -   (a) one or more esters having the structure

[X]_(m)R₅

-   -   wherein    -   X is an ester group of the formula R₆C(O)O;    -   R₆ is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with a hydroxyl group or C1 to C4 alkoxy        group, wherein R₆ optionally comprises one or more ether        linkages where R₆ is C2 to C7;    -   R₅ is a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety        optionally substituted with a hydroxyl group, wherein each        carbon atom in R₅ individually comprises no more than one        hydroxyl group or no more than one ester group, and wherein R₅        optionally comprises one or more ether linkages;    -   m is 1 to the number of carbon atoms in R₅,    -   said one or more esters having a solubility in water of at least        5 ppm at 25° C.; or    -   (b) one or more glycerides having the structure

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O); or    -   (c) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10; or    -   (d) one or more acetylated monosaccharides, acetylated        disaccharides, or acetylated polysaccharides; or    -   (e) any combination of (a) through (d).

Examples of said carboxylic acid ester substrate may include monoacetin;triacetin; monopropionin; dipropionin; tripropionin; monobutyrin;dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate;acetylated xylan; acetylated xylan fragments;β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal;tri-O-acetyl-glucal; propylene glycol diacetate; ethylene glycoldiacetate; monoesters or diesters of 1,2-ethanediol; 1,2-propanediol;1,3-propanediol; 1,2-butanediol; 1,3-butanediol; 2,3-butanediol;1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol; 1,6-pentanediol,1,2-hexanediol; 2,5-hexanediol; 1,6-hexanediol; or any combinationthereof.

As used herein, the term “peracid” is synonymous with peroxyacid,peroxycarboxylic acid, peroxy acid, percarboxylic acid and peroxoicacid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate,1,2,3-triacetoxypropane; 1,2,3-propanetriol triacetate and all othersynonyms of CAS Registry Number 102-76-1

As used herein, the term “monobutyrin” is synonymous with glycerolmonobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term “dibutyrin” is synonymous with glyceroldibutyrate and glyceryl dibutyrate.

As used herein, the term “tributyrin” is synonymous with glyceroltributyrate, 1,2,3-tributyrylglycerol, and all other synonyms of CASRegistry Number 60-01-5.

As used herein, the term “monopropionin” is synonymous with glycerolmonopropionate, glycerin monopropionate, and glyceryl monopropionate.

As used herein, the term “dipropionin” is synonymous with glyceroldipropionate and glyceryl dipropionate.

As used herein, the term “tripropionin” is synonymous with glyceryltripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, andall other synonyms of CAS Registry Number 139-45-7.

As used herein, the term “ethyl acetate” is synonymous with aceticether, acetoxyethane, ethyl ethanoate, acetic acid ethyl ester, ethanoicacid ethyl ester, ethyl acetic ester and all other synonyms of CASRegistry Number 141-78-6.

As used herein, the term “ethyl lactate” is synonymous with lactic acidethyl ester and all other synonyms of CAS Registry Number 97-64-3.

As used herein, the terms “acetylated sugar” and “acetylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acetylgroup. Examples include, but are not limited to, glucose pentaacetate,xylose tetraacetate, acetylated xylan, acetylated xylan fragments,13-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-glucal.

As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and“hydrocarbyl moiety” is meant a straight chain, branched or cyclicarrangement of carbon atoms connected by single, double, or triplecarbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety isa straight chain, branched or cyclic arrangement of carbon atomsconnected by single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms.

As used herein, the terms “monoesters” and “diesters” of 1,2-ethanediol;1,2-propanediol; 1,3-propanediol; 1,2-butanediol; 1,3-butanediol;2,3-butanediol; 1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol;1,6-pentanediol; 1,2-hexanediol; 2,5-hexanediol; 1,6-hexanediol; andmixtures thereof, refer to said compounds comprising at least one estergroup of the formula RC(O)O, wherein R is a C1 to C7 linear hydrocarbylmoiety. In one embodiment, the carboxylic acid ester substrate isselected from the group consisting of propylene glycol diacetate (PGDA),ethylene glycol diacetate (EDGA), and mixtures thereof.

As used herein, the term “propylene glycol diacetate” is synonymous with1,2-diacetoxypropane, propylene diacetate, 1,2-propanediol diacetate,and all other synonyms of CAS Registry Number 623-84-7.

As used herein, the term “ethylene glycol diacetate” is synonymous with1,2-diacetoxyethane, ethylene diacetate, glycol diacetate, and all othersynonyms of CAS Registry Number 111-55-7.

As used herein, the terms “suitable enzymatic reaction mixture”,“components suitable for in situ generation of a peracid”, “suitablereaction components”, and “suitable aqueous reaction mixture” refer tothe materials and water in which the reactants and enzyme catalyst comeinto contact. The components of the suitable aqueous reaction mixtureare provided herein and those skilled in the art appreciate the range ofcomponent variations suitable for this process. In one embodiment, thesuitable enzymatic reaction mixture produces peracid in situ uponcombining the reaction components. As such, the reaction components maybe provided as a multicomponent system wherein one or more of thereaction components remains separated until use. In another embodiment,the reaction components are first combined to form an aqueous solutionof peracid which is subsequently contacted with the surface to bedisinfected and/or bleached. The design of systems and means forseparating and combining multiple active components are known in the artand generally will depend upon the physical form of the individualreaction components. For example, multiple active fluids (liquid-liquid)systems typically use multi-chamber dispenser bottles or two-phasesystems (e.g., U.S. Patent Application Publication No. 2005/0139608;U.S. Pat. No. 5,398,846; U.S. Pat. No. 5,624,634; U.S. Pat. No.6,391,840; E.P. Patent 0807156B1; U.S. Patent Application PublicationNo. 2005/0008526; and PCT Publication No. WO 00/61713) such as found insome bleaching applications wherein the desired bleaching agent isproduced upon mixing the reactive fluids. Other forms of multi-componentsystems used to generate peracid may include, but are not limited to,those designed for one or more solid components or combinations ofsolid-liquid components, such as powders (e.g., U.S. Pat. No.5,116,575), multi-layered tablets (e.g., U.S. Pat. No. 6,210,639), waterdissolvable packets having multiple compartments (e.g., U.S. Pat. No.6,995,125) and solid agglomerates that react upon the addition of water(e.g., U.S. Pat. No. 6,319,888). In one embodiment, a multicomponentformulation is provided as two individual components whereby an aqueoussolution comprising a peroxycarboxylic acid is generated upon combiningthe two components. In another embodiment, a multi-component formulationis provided comprising:

-   -   a) a first component comprising:        -   i) an enzyme powder as disclosed herein; and        -   ii) a carboxylic acid ester, said first component optionally            comprising a further ingredient selected from the group            consisting of an inorganic or organic buffer, a corrosion            inhibitor, a wetting agent, and combinations thereof; and    -   b) a second component comprising a source of peroxygen and        water, said second component optionally comprising a hydrogen        peroxide stabilizer.

In another embodiment, the carboxylic acid ester in the first componentis selected from the group consisting of monoacetin, diacetin,triacetin, and combinations thereof. In another embodiment, thecarboxylic acid ester in the first component is an acetylatedsaccharide. In another embodiment, the enzyme catalyst in the firstcomponent is a particulate solid. In another embodiment, the firstreaction component is a solid tablet or powder.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with peroxide to form a peracid. Typically, inorganicperoxide is reacted with the selected substrate in the presence of acatalyst to produce the peracid. As used herein, the term “chemicalperhydrolysis” includes perhydrolysis reactions in which a substrate (aperacid precursor) is combined with a source of hydrogen peroxidewherein peracid is formed in the absence of an enzyme catalyst.

As used herein, the term “perhydrolase activity” refers to the catalystactivity per unit mass (for example, milligram) of protein, dry cellweight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmol of peracid product per minute at a specifiedtemperature.

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme having perhydrolysis activityand may be in the form of a whole microbial cell, permeabilizedmicrobial cell(s), one or more cell components of a microbial cellextract, partially purified enzyme, or purified enzyme. The enzymecatalyst may also be chemically modified (e.g., by pegylation or byreaction with cross-linking reagents). The perhydrolase catalyst mayalso be immobilized on a soluble or insoluble support using methodswell-known to those skilled in the art; see for example, Immobilizationof Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press,Totowa, N.J., USA; 1997. As described herein, all of the present enzymeshaving perhydrolysis activity are structurally members of thecarbohydrate family esterase family 7 (CE-7 family) of enzymes (seeCoutinho, P. M., Henrissat, B. “Carbohydrate-active enzymes: anintegrated database approach” in Recent Advances in CarbohydrateBioengineering, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.). TheCE-7 family of enzymes has been demonstrated to be particularlyeffective for producing peracids from a variety of carboxylic acid estersubstrates when combined with a source of peroxygen (See PCT publicationNo. WO2007/070609 and U.S. Patent Application Publication Nos.2008/0176299, 2008/176783, and 2009/0005590 to DiCosimo et al.; eachherein incorporated by reference in their entireties).

Members of the CE-7 family include cephalosporin C deacetylases (CAHs;E.C. 3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72). Membersof the CE-7 esterase family share a conserved signature motif (Vincentet al., J. Mol. Biol., 330:593-606 (2003)). Perhydrolases comprising theCE-7 signature motif and/or a substantially similar structure aresuitable for use in the present invention. Means to identifysubstantially similar biological molecules are well known in the art(e.g., sequence alignment protocols, nucleic acid hybridizations, and/orthe presence of a conserved signature motif). In one aspect, the presentperhydrolases include enzymes comprising the CE-7 signature motif and atleast 30%, preferably at least 33%, more preferably at least 40%, evenmore preferably at least 42%, even more preferably at least 50%, evenmore preferably at least 60%, even more preferably at least 70%, evenmore preferably at least 80%, even more preferably at least 90%, andmost preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% amino acid identity to the sequences provided herein. In a furtheraspect, the present perhydrolases include enzymes comprising the CE-7signature motif and at least 30%, preferably at least 33%, morepreferably at least 40%, even more preferably at least 42%, even morepreferably at least 50%, even more preferably at least 60%, even morepreferably at least 70%, even more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID NO:1.

As used herein, the term “enzyme powder” refers to the spray-driedproduct of an aqueous formulation comprising (1) at least one enzymestructurally classified as a CE-7 carbohydrate esterase that hasperhydrolysis activity, (2) at least one oligosaccharide excipient, andoptionally at least one surfactant. In some embodiments, the at leastone oligosaccharide excipient has a number average molecular weight ofat least about 1250 and a weight average molecular weight of at leastabout 9000. In one embodiment, the aqueous formulation further comprisesat least one buffer.

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refer to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., (1995) Appl. Env.Microbiol. 61(6):2224-2229). Several cephalosporin C deacetylases areprovided herein having significant perhydrolysis activity.

As used herein, “acetyl xylan esterases” refers to an enzyme (E.C.3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylansand other acetylated saccharides. As illustrated herein, several enzymesclassified as acetyl xylan esterases are provided having significantperhydrolysis activity.

As used herein, the term “Bacillus subtilis ATCC® 31954™” refers to abacterial cell deposited to the American Type Culture Collection (ATCC®)having international depository accession number ATCC® 31954™. Bacillussubtilis ATCC® 31954™ has been reported to have an ester hydrolase(“diacetinase”) activity capable of hydrolyzing glycerol esters having 2to 8 carbon acyl groups, especially diacetin (U.S. Pat. No. 4,444,886;herein incorporated by reference in its entirety). As described herein,an enzyme having significant perhydrolase activity has been isolatedfrom B. subtilis ATCC® 31954™ and is provided as SEQ ID NO:1. The aminoacid sequence of the isolated enzyme has 100% amino acid identity to thecephalosporin C deacetylase provided by GENBANK® Accession No.BAA01729.1 (Mitsushima et al., supra).

As used herein, the term “Bacillus subtilis ATCC® 29233™” refers to astrain of Bacillus subtilis deposited to the American Type CultureCollection (ATCC®) having international depository accession numberATCC® 29233™. As described herein, an enzyme having significantperhydrolase activity has been isolated and sequenced from B. subtilisATCC® 29233™ and is provided as SEQ ID NO:12.

As used herein, the term “Clostridium thermocellum ATCC® 27405™” refersto a strain of Clostridium thermocellum deposited to the American TypeCulture Collection (ATCC®) having international depository accessionnumber ATCC® 27405™. The amino acid sequence of the enzyme havingperhydrolase activity from C. thermocellum ATCC® 27405™ is provided asSEQ ID NO:5.

As used herein, the term “Bacillus subtilis ATCC® 6633™” refers to abacterial cell deposited to the American Type Culture Collection (ATCC®)having international depository accession number ATCC® 6633™. Bacillussubtilis ATCC® 6633™ has been reported to have cephalosporinacetylhydrolase activity (U.S. Pat. No. 6,465,233). The amino acidsequence of the enzyme having perhydrolase activity from B. subtilisATCC® 6633™ is provided as SEQ ID NO:2.

As used herein, the term “Bacillus licheniformis ATCC® 14580™” refers toa bacterial cell deposited to the American Type Culture Collection(ATCC®) having international depository accession number ATCC® 14580™.Bacillus licheniformis ATCC® 14580™ has been reported to havecephalosporin acetylhydrolase activity. The amino acid sequence of theenzyme having perhydrolase activity from B. licheniformis ATCC® 14580™is provided as SEQ ID NO:3.

As used herein, the term “Bacillus pumilus PS213” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®AJ249957).

The amino acid sequence of the enzyme having perhydrolase activity fromBacillus pumilus PS213 is provided as SEQ ID NO:4.

As used herein, the term “Thermotoga neapolitana” refers to a strain ofThermotoga neapolitana reported to have acetyl xylan esterase activity(GENBANK® AAB70869). The amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga neapolitana is provided as SEQ IDNO: 6.

As used herein, the term “Thermotoga maritima MSB8” refers to abacterial cell reported to have acetyl xylan esterase activity (GENBANK®NP_(—)227893.1). The amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga maritima MSB8 is provided as SEQID NO: 7.

As used herein, the term “Bacillus clausii KSM-K16” refers to abacterial cell reported to have cephalosporin-C deacetylase activity(GENBANK® YP_(—)175265). The amino acid sequence of the enzyme havingperhydrolase activity from Bacillus clausii KSM-K16 is provided as SEQID NO: 11.

As used herein, the term “Thermoanearobacterium saccharolyticum” refersto a bacterial strain reported to have acetyl xylan esterase activity(GENBANK® S41858). The amino acid sequence of the enzyme havingperhydrolase activity from Thermoanearobacterium saccharolyticum isprovided as SEQ ID NO: 13.

As used herein, the term “Thermotoga lettingae” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®CP000812). The deduced amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga lettingae is provided as SEQ IDNO: 14.

As used herein, the term “Thermotoga petrophila” refers to a bacterialcell reported to have acetyl xylan esterase activity (GENBANK®CP000702). The deduced amino acid sequence of the enzyme havingperhydrolase activity from Thermotoga lettingae is provided as SEQ IDNO: 15.

As used herein, the term “Thermotoga sp. RQ2” refers to a bacterial cellreported to have acetyl xylan esterase activity (GENBANK® CP000969). Twodifferent acetyl xylan esterases have been identified from Thermotogasp. RQ2 and are referred to herein as “RQ2(a)” (the deduced amino acidsequence provided as SEQ ID NO: 16) and “RQ2(b)” (the deduced amino acidsequence provided as SEQ ID NO: 17).

As used herein, an “isolated nucleic acid molecule” and “isolatednucleic acid fragment” will be used interchangeably and refer to apolymer of RNA or DNA that is single- or double-stranded, optionallycontaining synthetic, non-natural or altered nucleotide bases. Anisolated nucleic acid molecule in the form of a polymer of DNA may becomprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid or asdefined herein Xaa X

As used herein, “substantially similar” refers to nucleic acid moleculeswherein changes in one or more nucleotide bases results in the addition,substitution, or deletion of one or more amino acids, but does notaffect the functional properties (i.e. perhydrolytic activity) of theprotein encoded by the DNA sequence. As used herein, “substantiallysimilar” also refers to an enzyme having an amino acid sequence that isat least 30%, preferably at least 33%, more preferably at least 40%,more preferably at least 50%, even more preferably at least 60%, evenmore preferably at least 70%, even more preferably at least 80%, yeteven more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to the sequences reported herein wherein theresulting enzyme retains the present functional properties (i.e.,perhydrolytic activity). “Substantially similar” may also refer to anenzyme having perhydrolytic activity encoded by nucleic acid moleculesthat hybridize under stringent conditions to the nucleic acid moleculesreported herein. It is therefore understood that the inventionencompasses more than the specific exemplary sequences.

For example, it is well known in the art that alterations in a genewhich result in the production of a chemically equivalent amino acid ata given site, but do not affect the functional properties of the encodedprotein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

-   -   1. Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly);    -   2. Polar, negatively charged residues and their amides: Asp,        Asn, Glu, Gln;    -   3. Polar, positively charged residues: His, Arg, Lys;    -   4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys);        and    -   5. Large aromatic residues: Phe, Tyr, and Trp.        Thus, a codon for the amino acid alanine, a hydrophobic amino        acid, may be substituted by a codon encoding another less        hydrophobic residue (such as glycine) or a more hydrophobic        residue (such as valine, leucine, or isoleucine). Similarly,        changes which result in substitution of one negatively charged        residue for another (such as aspartic acid for glutamic acid) or        one positively charged residue for another (such as lysine for        arginine) can also be expected to produce a functionally        equivalent product. In many cases, nucleotide changes which        result in alteration of the N-terminal and C-terminal portions        of the protein molecule would also not be expected to alter the        activity of the protein.

Each of the proposed modifications is well within the routine skill inthe art, as is determination of retention of biological activity of theencoded products. Moreover, the skilled artisan recognizes thatsubstantially similar sequences are encompassed by the presentinvention. In one embodiment, substantially similar sequences aredefined by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS, 65° C.) with the sequences exemplified herein.

As used herein, a nucleic acid molecule is “hybridizable” to anothernucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when asingle strand of the first molecule can anneal to the other moleculeunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known and exemplified inSambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(2001). The conditions of temperature and ionic strength determine the“stringency” of the hybridization. Stringency conditions can be adjustedto screen for moderately similar molecules, such as homologous sequencesfrom distantly related organisms, to highly similar molecules, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes typically determine stringency conditions. Oneset of preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A more preferred set of conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of stringent hybridizationconditions is 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDSfollowed by a final wash of 0.1×SSC, 0.1% SDS, 65° C. with the sequencesexemplified herein. In a further embodiment, the present compositionsand methods employ an enzyme having perhydrolase activity encoded byisolated nucleic acid molecule that hybridizes under stringentconditions to a nucleic acid molecule encoding a polypeptide havingperhydrolysis activity, said polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13; SEQ ID NO: 14; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (Sambrook andRussell, supra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrookand Russell, supra). In one aspect, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Preferably, a minimumlength for a hybridizable nucleic acid is at least about 15 nucleotidesin length, more preferably at least about 20 nucleotides in length, evenmore preferably at least 30 nucleotides in length, even more preferablyat least 300 nucleotides in length, and most preferably at least 800nucleotides in length. Furthermore, the skilled artisan will recognizethat the temperature and wash solution salt concentration may beadjusted as necessary according to factors such as length of the probe.

As used herein, the term “percent identity” is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to, methods described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, NY (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994);Sequence Analysis in Molecular Biology (von Heinje, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Stockton Press, NY (1991). Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.), the AlignX program of Vector NTI v.7.0 (Informax, Inc., Bethesda, Md.), or the EMBOSS Open Software Suite(EMBL-EBI; Rice et al., Trends in Genetics 16, (6):276-277 (2000)).Multiple alignment of the sequences can be performed using the Clustalmethod (e.g., CLUSTALW; for example version 1.83) of alignment (Higginsand Sharp, CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res.22:4673-4680 (1994); and Chema et al., Nucleic Acids Res 31(13):3497-500 (2003)), available from the European Molecular BiologyLaboratory via the European Bioinformatics Institute) with the defaultparameters. Suitable parameters for CLUSTALW protein alignments includeGAP Existence penalty=15, GAP extension=0.2, matrix=Gonnet (e.g.Gonnet250), protein ENDGAP=−1, Protein GAPDIST=4, and KTUPLE=1. In oneembodiment, a fast or slow alignment is used with the default settingswhere a slow alignment is preferred. Alternatively, the parameters usingthe CLUSTALW method (version 1.83) may be modified to also use KTUPLE=1,GAP PENALTY=10, GAP extension=1, matrix=BLOSUM (e.g. BLOSUM64),WINDOW=5, and TOP DIAGONALS SAVED=5.

In one aspect, suitable isolated nucleic acid molecules encode apolypeptide having an amino acid sequence that is at least about 30%,preferably at least 33%, preferably at least 40%, preferably at least50%, preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, even more preferably at least 85%, even morepreferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequences reported herein. Suitable nucleicacid molecules not only have the above homologies, but also typicallyencode a polypeptide having about 300 to about 340 amino acids, morepreferably about 310 to about 330 amino acids, and most preferably about318 amino acids.

As used herein, the terms “signature motif”, “CE-7 signature motif”, and“diagnostic motif” refer to conserved structures shared among a familyof enzymes having a defined activity. The signature motif can be used todefine and/or identify the family of structurally related enzymes havingsimilar enzymatic activity for a defined family of substrates. Thesignature motif can be a single contiguous amino acid sequence or acollection of discontiguous, conserved motifs that together form thesignature motif. Typically, the conserved motif(s) is represented by anamino acid sequence. As described herein, the present enzymes havingperhydrolysis activity (“perhydrolases”) belong to the family of CE-7carbohydrate esterases (DiCosimo et al., supra). As used herein, thephrase “enzyme is structurally classified as a CE-7 enzyme” or “CE-7perhydrolase” will be used to refer to enzymes having perhydrolysisactivity which are structurally classified as a CE-7 carbohydrateesterase. This family of enzymes can be defined by the presence of asignature motif (Vincent et al., supra). As defined herein, thesignature motif for CE-7 esterases comprises three conserved motifs(residue position numbering relative to reference sequence SEQ ID NO:1):

-   -   a) Arg118-Gly119-Gln120;    -   b) Gly179-Xaa180-Ser181-Gln182-Gly183; and    -   c) His298-Glu299.        Typically, the Xaa at amino acid residue position 180 is        glycine, alanine, proline, tryptophan, or threonine. Two of the        three amino acid residues belonging to the catalytic triad are        in bold. In one embodiment, the Xaa at amino acid residue        position 180 is selected from the group consisting of glycine,        alanine, proline, tryptophan, and threonine.

Further analysis of the conserved motifs within the CE-7 carbohydrateesterase family indicates the presence of an additional conserved motif(LXD at amino acid positions 267-269 of SEQ ID NO: 1) that may be usedto further define a perhydrolase belonging to the CE-7 carbohydrateesterase family. In a further embodiment, the signature motif definedabove includes a fourth conserved motif defined as:

Leu267-Xaa268-Asp269.

The Xaa at amino acid residue position 268 is typically isoleucine,valine, or methionine. The fourth motif includes the aspartic acidresidue (bold) belonging to the catalytic triad (Ser181-Asp269-His298).

A number of well-known global alignment algorithms may be used to aligntwo or more amino acid sequences representing enzymes havingperhydrolase activity to determine if the enzyme is comprised of thepresent signature motif. The aligned sequence(s) are compared to thereference sequence (SEQ ID NO:1) to determine the existence of thesignature motif. In one embodiment, a CLUSTAL alignment (such asCLUSTALW) using a reference amino acid sequence (as used herein theperhydrolase sequence (SEQ ID NO:1) from the Bacillus subtilis ATCC®31954™) is used to identify perhydrolases belonging to the CE-7 esterasefamily. The relative numbering of the conserved amino acid residues isbased on the residue numbering of the reference amino acid sequence toaccount for small insertions or deletions (for example, five amino acidsof less) within the aligned sequence.

Examples of other suitable algorithms that may be used to identifysequences comprising the present signature motif (when compared to thereference sequence) include, but are not limited to, Needleman andWunsch (J. Mol. Biol. 48, 443-453 (1970); a global alignment tool) andSmith-Waterman (J. Mol. Biol. 147:195-197 (1981); a local alignmenttool). In one embodiment, a Smith-Waterman alignment is implementedusing default parameters. An example of suitable default parametersinclude the use of a BLOSUM62 scoring matrix with GAP open penalty=10and a GAP extension penalty=0.5.

A comparison of the overall percent identity among perhydrolasesexemplified herein indicates that enzymes having as little as 33%identity to SEQ ID NO:1 (while retaining the signature motif) exhibitsignificant perhydrolase activity and are structurally classified asCE-7 carbohydrate esterases. In one embodiment, suitable perhydrolasesinclude enzymes comprising the CE-7 signature motif and at least 30%,preferably at least 33%, more preferably at least 40%, even morepreferably at least 42%, even more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, even more preferably at least 90%, and mostpreferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%amino acid identity to SEQ ID NO: 1.

Alternatively, a contiguous amino acid sequence comprising the regionencompassing the conserved motifs may also be used to identify CE-7family members.

As used herein, “codon degeneracy” refers to the nature of the geneticcode permitting variation of the nucleotide sequence without affectingthe amino acid sequence of an encoded polypeptide. Accordingly, thepresent invention relates to any nucleic acid molecule that encodes allor a substantial portion of the amino acid sequences encoding thepresent microbial polypeptide. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing agene for improved expression in a host cell, it is desirable to designthe gene such that its frequency of codon usage approaches the frequencyof preferred codon usage of the host cell.

As used herein, the term “codon optimized”, as it refers to genes orcoding regions of nucleic acid molecules for transformation of varioushosts, refers to the alteration of codons in the gene or coding regionsof the nucleic acid molecules to reflect the typical codon usage of thehost organism without altering the polypeptide for which the DNA codes.

As used herein, “synthetic genes” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form gene segments that are then enzymatically assembled toconstruct the entire gene. “Chemically synthesized”, as pertaining to aDNA sequence, means that the component nucleotides were assembled invitro. Manual chemical synthesis of DNA may be accomplished usingwell-established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the genes can be tailored for optimal gene expression basedon optimization of nucleotide sequences to reflect the codon bias of thehost cell. The skilled artisan appreciates the likelihood of successfulgene expression if codon usage is biased towards those codons favored bythe host. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

As used herein, “gene” refers to a nucleic acid molecule that expressesa specific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different from that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein, “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Suitable regulatory sequences”refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, RNAprocessing site, effector binding site and stem-loop structure.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene at different stages of development, or in responseto different environmental or physiological conditions. Promoters thatcause a gene to be expressed at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of different lengths may haveidentical promoter activity.

As used herein, the “3′ non-coding sequences” refer to DNA sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences (normally limited to eukaryotes) and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts(normally limited to eukaryotes) to the 3′ end of the mRNA precursor.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid molecule so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence, i.e., that the coding sequenceis under the transcriptional control of the promoter. Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

As used herein, the term “expression” refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid molecule of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein, “transformation” refers to the transfer of a nucleicacid molecule into the genome of a host organism, resulting ingenetically stable inheritance. In the present invention, the hostcell's genome includes chromosomal and extrachromosomal (e.g. plasmid)genes. Host organisms containing the transformed nucleic acid moleculesare referred to as “transgenic” or “recombinant” or “transformed”organisms.

As used herein, the terms “plasmid”, “vector” and “cassette” refer to anextrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein, the term “sequence analysis software” refers to anycomputer algorithm or software program that is useful for the analysisof nucleotide or amino acid sequences. “Sequence analysis software” maybe commercially available or independently developed. Typical sequenceanalysis software will include, but is not limited to, the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), CLUSTALW (for example, version 1.83; Thompson et al.,Nucleic Acids Research, 22(22):4673-4680 (1994), and the FASTA programincorporating the Smith-Waterman algorithm (W. R. Pearson, Comput.Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.),Vector NTI (Informax, Bethesda, Md.) and Sequencher v. 4.05. Within thecontext of this application it will be understood that where sequenceanalysis software is used for analysis, that the results of the analysiswill be based on the “default values” of the program referenced, unlessotherwise specified. As used herein “default values” will mean any setof values or parameters set by the software manufacturer that originallyload with the software when first initialized.

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to, microorganisms, spores, viruses, prions, and mixturesthereof. The process produces an efficacious concentration of at leastone percarboxylic acid useful to reduce and/or eliminate the presence ofthe viable biological contaminants. In a preferred embodiment, thebiological contaminant is a viable pathogenic microorganism.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to an agent thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. Typically, disinfectants are used to treatinanimate objects or surfaces. As used herein, the term “disinfection”refers to the act or process of disinfecting. As used herein, the term“antiseptic” refers to a chemical agent that inhibits the growth ofdisease-carrying microorganisms. In one aspect, the biologicalcontaminants are pathogenic microorganisms.

As used herein, the term “sanitary” means of or relating to therestoration or preservation of health, typically by removing, preventingor controlling an agent that may be injurious to health. As used herein,the term “sanitize” means to make sanitary. As used herein, the term“sanitizer” refers to a sanitizing agent. As used herein the term“sanitization” refers to the act or process of sanitizing.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses, and is synonymous with “viricide”. An agent thatexhibits the ability to inhibit or destroy viruses is described ashaving “virucidal” activity. Peracids can have virucidal activity.Typical alternative virucides known in the art which may be suitable foruse with the present invention include, for example, alcohols, ethers,chloroform, formaldehyde, phenols, beta propiolactone, iodine, chlorine,mercury salts, hydroxylamine, ethylene oxide, ethylene glycol,quaternary ammonium compounds, enzymes, and detergents.

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peracids can have biocidalactivity. Typical alternative biocides known in the art, which may besuitable for use in the present invention include, for example,chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone,acrolein, amines, chlorinated phenolics, copper salts, organo-sulphurcompounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viablemicroorganisms after treatment is at least a 3-log reduction, morepreferably at least a 4-log reduction, and most preferably at least a5-log reduction. In another aspect, the minimum biocidal concentrationis at least a 6-log reduction in viable microbial cells.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to, hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates. As described herein, the concentration ofthe hydrogen peroxide provided by the peroxygen compound in the aqueousreaction formulation is initially at least 1 mM or more upon combiningthe reaction components. In one embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is at least 10 mM. Inanother embodiment, the hydrogen peroxide concentration in the aqueousreaction formulation is at least 100 mM. In another embodiment, thehydrogen peroxide concentration in the aqueous reaction formulation isat least 200 mM. In another embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is 500 mM or more. Inyet another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is 1000 mM or more. The molar ratio of thehydrogen peroxide to enzyme substrate, e.g. triglyceride,(H₂O₂:substrate) in the aqueous reaction formulation may be from about0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5to 5.

By “oligosaccharide” is meant compounds containing between 2 and atleast 24 monosaccharide units linked by glycosidic linkages. The term“monosaccharide” refers to a compound of empirical formula (CH₂O)_(n),where n≧13, the carbon skeleton is unbranched, each carbon atom exceptone contains a hydroxyl group, and the remaining carbon atom is analdehyde or ketone at carbon atom 2. The term “monosaccharide” alsorefers to intracellular cyclic hemiacetal or hemiketal forms.

As used herein, the term “excipient” refers to an inactive substanceused to stabilize the active ingredient in a formulation. Excipients arealso sometimes used to bulk up formulations that contain activeingredients. As described herein, the “active ingredient” is an enzymecatalyst comprising at least one enzyme having perhydrolysis activity.In one embodiment, the active ingredient is at least one CE-7carbohydrate esterase having perhydrolysis activity.

As used herein, the term “oligosaccharide excipient” means anoligosaccharide that, when added to an aqueous enzyme solution, improvesrecovery/retention of active enzyme (i.e., perhydrolase activity) afterspray drying and/or improves storage stability of the resultingspray-dried enzyme powder or a formulation of the enzyme powder and acarboxylic acid ester. In one embodiment, the addition of theoligosaccharide excipient prior to spray drying improves the storagestability of the enzyme when stored in the carboxylic acid ester (i.e.,a storage mixture substantially free of water). The carboxylic acidester may contain a very low concentration of water, for example,triacetin typically has between 180 ppm and 300 ppm of water. As usedherein, the phrase “substantially free of water” will refer to aconcentration of water in a mixture of the enzyme powder and thecarboxylic acid ester that does not adversely impact the storagestability of enzyme powder when present in the carboxylic acid ester. Ina further embodiment, “substantially free of water” may mean less than2000 ppm, preferably less than 1000 ppm, more preferably less than 500ppm, and even more preferably less than 250 ppm of water in theformulation comprising the enzyme powder and the carboxylic acid ester.

Enzyme Powder

One aspect is for an enzyme powder comprising a spray-dried formulationof at least one enzyme structurally classified as a CE-7 enzyme andhaving perhydrolysis activity, at least one oligosaccharide excipient,and optionally at least one surfactant. In some embodiments, the atleast one oligosaccharide excipient has a number average molecularweight of at least about 1250 and a weight average molecular weight ofat least about 9000.

The at least one enzyme can be any of the CE-7 carbohydrate esterasesdescribed herein or can be any of the CE-7 carbohydrate esterasesdescribed in co-owned, copending Published U.S. Patent Application Nos.2008/0176299 and 2009/0005590 (each incorporated herein by reference inits entirety). In some embodiments, the at least one enzyme is selectedfrom the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, and 25.

The at least one enzyme is present in the spray-dried formulation in anamount in a range of from about 5 wt % to about 75 wt % based on the dryweight of the spray-dried formulation. A preferred wt % range of enzymein the spray-dried formulation is from about 10 wt % to 50 wt %, and amore preferred wt % range of enzyme in the spray-dried formulation isfrom about 20 wt % to 33 wt %

The spray-dried formulation further comprises at least oneoligosaccharide excipient. In some embodiments, the at least oneoligosaccharide excipient has a number average molecular weight of atleast about 1250 and a weight average molecular weight of at least about9000. In some embodiments, the oligosaccharide excipient has a numberaverage molecular weight of at least about 1700 and a weight averagemolecular weight of at least about 15000. Specific oligosaccharidesuseful in the present invention include, but are not limited to,maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan,raffinose, stachyose, pectin, inulin, levan, graminan, amylopectin,sucrose, lactulose, lactose, maltose, trehalose, cellobiose,nigerotriose, maltotriose, melezitose, maltotriulose, raffinose,kestose, and mixtures thereof. Oligosaccharide-based excipients usefulin the present invention include, but are not limited to, water-solublenon-ionic cellulose ethers, such as hydroxymethyl-cellulose andhydroxypropylmethylcellulose, and mixtures thereof.

The excipient is present in the formulation in an amount in a range offrom about 95 wt % to about 25 wt % based on the dry weight of thespray-dried formulation. A preferred wt % range of excipient in thespray-dried formulation is from about 90 wt % to 50 wt %, and a morepreferred wt % range of excipient in the spray-dried formulation is fromabout 80 wt % to 67 wt %.

In some embodiments, the formulation further comprises at least onesurfactant. Useful surfactants include, but are not limited to, ionicand nonionic surfactants or wetting agents, such as ethoxylated castoroil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitanfatty acid esters, poloxamers, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene derivatives, monoglycerides or ethoxylatedderivatives thereof, diglycerides or polyoxyethylene derivativesthereof, sodium docusate, sodium laurylsulfate, cholic acid orderivatives thereof, lecithins, phospholipids, block copolymers ofethylene glycol and propylene glycol, and non-ionic organosilicones.Preferably, the surfactant is a polyoxyethylene sorbitan fatty ester,with polysorbate 80 being more preferred.

When part of the formulation, the surfactant is present in an amount ina range of from about 5 wt % to 0.1 wt % based on the weight of proteinpresent in the spray dried formulation, preferably from about 2 wt % to0.5 wt % based on the weight of protein present in the spray driedformulation.

The spray dried formulation may additionally comprise one or morebuffers (e.g., sodium and/or potassium salts of bicarbonate, citrate,acetate, phosphate, pyrophosphate, methylphosphonate, succinate, malate,fumarate, tartrate, or maleate), and an enzyme stabilizer (such asethylenediaminetetraacetic acid, (1-hydroxyethylidene)bisphosphonicacid).

Spray drying of the formulation of at least one enzyme, at least oneoligosaccharide excipient, and optionally at least one surfactant iscarried out, for example, as described generally in the Spray DryingHandbook, 5^(th) ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y.(1991), and in PCT Patent Publication Nos. WO 97/41833 (1997) and WO96/32149 (1996) to Platz, R., et al.

In general spray drying consists of bringing together a highly dispersedliquid and a sufficient volume of hot air to produce evaporation anddrying of the liquid droplets. Typically the feed is sprayed into acurrent of warm filtered air that evaporates the solvent and conveys thedried product to a collector. The spent air is then exhausted with thesolvent. Those skilled in the art will appreciate that several differenttypes of apparatus may be used to provide the desired product. Forexample, commercial spray dryers manufactured by Buchi Ltd. (Postfach,Switzerland) or GEA Niro Corp. (Copenhagen, Denmark) will effectivelyproduce particles of desired size. It will further be appreciated thatthese spray dryers, and specifically their atomizers, may be modified orcustomized for specialized applications, such as the simultaneousspraying of two solutions using a double nozzle technique. Morespecifically, a water-in-oil emulsion can be atomized from one nozzleand a solution containing an anti-adherent such as mannitol can beco-atomized from a second nozzle. In other cases it may be desirable topush the feed solution though a custom designed nozzle using a highpressure liquid chromatography (HPLC) pump. Provided thatmicrostructures comprising the correct morphology and/or composition areproduced the choice of apparatus is not critical and would be apparentto the skilled artisan in view of the teachings herein.

The temperature of both the inlet and outlet of the gas used to dry thesprayed material is such that it does not cause degradation of theenzyme in the sprayed material. Such temperatures are typicallydetermined experimentally, although generally, the inlet temperaturewill range from about 50° C. to about 225° C., while the outlettemperature will range from about 30° C. to about 150° C. Preferredparameters include atomization pressures ranging from about 20-150 psi(0.14 MPa-1.03 MPa), and preferably from about 30-40 to 100 psi(0.21-0.28 MPa to 0.69 MPa). Typically the atomization pressure employedwill be one of the following (MPa) 0.14, 0.21, 0.28, 0.34, 0.41, 0.48,0.55, 0.62, 0.69, 0.76, 0.83 or above.

The spray-dried enzyme powder or a formulation of the spray-dried enzymepowder in carboxylic acid ester substantially retains its enzymaticactivity for an extended period of time when stored at ambienttemperature. The spray-dried enzyme powder or a formulation of thespray-dried enzyme powder in carboxylic acid ester substantially retainsits enzymatic activity at elevated temperatures for short periods oftime. In one embodiment, “substantially retains its enzymatic activity”is meant that the spray-dried enzyme powder or a formulation of thespray-dried enzyme powder in carboxylic acid ester retains at leastabout 75 percent of the enzyme activity of the enzyme in the spray-driedenzyme powder or a formulation of the spray-dried enzyme powder after anextended storage period at ambient temperature and/or after a shortstorage period at an elevated temperature (above ambient temperature) ina formulation comprised of a carboxylic acid ester and the enzyme powderas compared to the initial enzyme activity of the enzyme powder prior tothe preparation of a formulation comprised of the carboxylic acid esterand the enzyme powder. The extended storage period is a period of timeof from about one year to about two years at ambient temperature. In oneembodiment, the short storage period is at an elevated temperature for aperiod of time of from when the formulation comprised of a carboxylicacid ester and the enzyme powder is produced at 40° C. to about eightweeks at 40° C. In another embodiment, the elevated temperature is in arange of from about 30° C. to about 52° C. In a preferred embodiment,the elevated temperature is in a range of from about 30° C. to about 40°C.

In some embodiments, the spray-dried enzyme powder has at least 75percent of the enzyme activity of the at least one enzyme after eightweeks storage at 40° C. in a formulation comprised of a carboxylic acidester and the enzyme powder as compared to the initial enzyme activityof the enzyme powder prior to the preparation of a formulation comprisedof the carboxylic acid ester and the enzyme powder at 40° C. In otherembodiments, the enzyme powder has at least 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 percent of the enzyme activity of the at least one enzyme aftereight weeks storage at 40° C. in a formulation comprised of a carboxylicacid ester and the enzyme powder as compared to the initial enzymeactivity of the enzyme powder prior to the preparation of a formulationcomprised of the carboxylic acid ester and the enzyme powder at 40° C.Preferably, perhydrolysis activity is measured as described in Example8-13, infra, but any method of measuring perhydrolysis activity can beused in the practice of the present invention.

In some embodiments, further improvement in enzyme activity over thestated periods of time can be achieved by adding a buffer having abuffering capacity in a pH range of from about 5.5 to about 9.5 to theformulation comprised of the carboxylic acid ester and the spray-driedenzyme powder. Suitable buffer for use in the formulation may include,but is not limited to, sodium salt, potassium salt, or mixtures ofsodium or potassium salts of bicarbonate, pyrophosphate, phosphate,methylphosphonate, citrate, acetate, malate, fumarate, tartrate maleateor succinate. Preferred buffers for use in the formulation comprised ofthe carboxylic acid ester and the spray-dried enzyme powder include thesodium salt, potassium salt, or mixtures of sodium or potassium salts ofbicarbonate, phosphate, methylphosphonate, or citrate.

In embodiments where a buffer is present in the carboxylic acid esterand enzyme powder formulation, the buffer may be present in an amount ina range of from about 0.01 wt % to about 50 wt % based on the weight ofcarboxylic acid ester in the formulation comprised of carboxylic acidester and enzyme powder. The buffer may be present in a more preferredrange of from about 0.10% to about 10% based on the weight of carboxylicacid ester in the formulation comprised of carboxylic acid ester andenzyme powder. Further, in these embodiments, the comparison betweenperhydrolysis activities of the enzyme is determined as between (a) anenzyme powder which retains at least 75 percent of the perhydrolysisactivity of the at least one enzyme after eight weeks storage at 40° C.in a formulation comprised of a carboxylic acid ester, a buffer having abuffering capacity in a pH range of from about 5.5 to about 9.5, and theenzyme powder and (b) the initial perhydrolysis activity of the enzymepowder prior to the preparation of a formulation comprised of thecarboxylic acid ester, the buffer having a buffering capacity in a pHrange of from about 5.5 to about 9.5, and the enzyme powder.

It is intended that the spray-dried enzyme powder be stored as aformulation in the organic compound that is a substrate for the at leastone enzyme, such as triacetin. In the absence of added hydrogenperoxide, triacetin is normally hydrolyzed in aqueous solution by a CE-7carbohydrate esterase to produce diacetin and acetic acid, and theproduction of acetic acid results in a decrease in the pH of thereaction mixture. One requirement for long term storage stability of theenzyme in triacetin is that there not be significant reaction of thetriacetin with any water that might be present in the triacetin; thespecification for water content in one commercial triacetin (supplied byTessenderlo Group, Brussels, Belgium) is 0.03 wt % water (300 ppm). Anyhydrolysis of triacetin that occurs during storage of the enzyme intriacetin would produce acetic acid, which could result in a decrease inactivity or inactivation of the perhydrolysis activity of the CE-7carbohydrate esterases; the perhydrolase activity of the CE-7carbohydrate esterases is typically inactivated at or below a pH of 5.0(see U.S. patent application Ser. No. 12/539,025 to DiCosimo, R., etal.). The oligosaccharide excipient selected for use in the presentapplication must provide stability of the enzyme in the organicsubstrate for the enzyme under conditions where acetic acid might begenerated due to the presence of low concentrations of water in theformulation.

Suitable Reaction Conditions for the Enzyme-Catalyzed Preparation ofPeracids from Carboxylic Acid Esters and Hydrogen Peroxide

In one aspect of the invention, a process is provided to produce anaqueous formulation comprising a peracid by reacting one or morecarboxylic acid esters with source of peroxygen (hydrogen peroxide,sodium perborate or sodium percarbonate) in the presence of an enzymecatalyst having perhydrolysis activity. In one embodiment, the enzymecatalyst comprises at least one enzyme having perhydrolysis activity,wherein said enzyme is structurally classified as a member of the CE-7carbohydrate esterase family (CE-7; see Coutinho, P. M., Henrissat, B.,supra). In another embodiment, the perhydrolase catalyst is structurallyclassified as a cephalosporin C deacetylase. In another embodiment, theperhydrolase catalyst is structurally classified as an acetyl xylanesterase.

In one embodiment, the perhydrolase catalyst comprises an enzyme havingperhydrolysis activity and a signature motif comprising:

-   -   a) an RGQ motif at amino acid residues 118-120;    -   b) a GXSQG motif at amino acid residues 179-183; and    -   c) an HE motif at amino acid residues 298-299 when aligned to        reference sequence SEQ ID NO:1 using CLUSTALW.

In a further embodiment, the signature motif additional comprises afourth conserved motif defined as an LXD motif at amino acid residues267-269 when aligned to reference sequence SEQ ID NO:1 using CLUSTALW.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving the present signature motif and at least 30% amino acid identityto SEQ ID NO:1.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving perhydrolase activity selected from the group consisting of SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19,20, 21, 22, 23, 24, and 25.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving at least 40% amino acid identity to a contiguous signature motifdefined as SEQ ID NO:18 wherein the conserved motifs described above(i.e., RGQ, GXSQG, and HE, and optionally, LXD) are conserved.

In another embodiment, the perhydrolase catalyst comprises an enzymehaving an amino acid sequence selected from the group consisting of SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19,20, 21, 22, 23, 24, and 25, wherein said enzyme may have one or moreadditions, deletions, or substitutions so long as the signature motif isconserved and perhydrolase activity is retained.

Suitable carboxylic acid ester substrates may include esters provided bythe following formula:

[X]_(m)R₅

wherein X=an ester group of the formula R₆C(O)O

-   -   R₆═C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with hydroxyl groups or C1 to C4 alkoxy        groups, wherein R₆ optionally comprises one or more ether        linkages for R₆═C2 to C7;    -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety        optionally substituted with hydroxyl groups; wherein each carbon        atom in R₅ individually comprises no more than one hydroxyl        group or no more than one ester group; wherein R₅ optionally        comprises one or more ether linkages;    -   m=1 to the number of carbon atoms in R₅; and    -   wherein said esters have solubility in water of at least 5 ppm        at 25° C.

In other embodiments, suitable substrates may also include esters of theformula:

wherein R₁═C1 to C7 straight chain or branched chain alkyl optionallysubstituted with a hydroxyl or a C1 to C4 alkoxy group and R₂═C1 to C10straight chain or branched chain alkyl, alkenyl, alkynyl, aryl,alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂—O)_(n)H or(CH₂CH(CH₃)—O)_(n)H and n=1 to 10.

In other embodiments, suitable carboxylic acid ester substrates mayinclude glycerides of the formula:

wherein R₁═C1 to C7 straight chain or branched chain alkyl optionallysubstituted with a hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄ areindividually H or R₁C(O).

In other embodiments, R₆ is C1 to C7 linear hydrocarbyl moiety,optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups,optionally comprising one or more ether linkages. In further preferredembodiments, R₆ is C2 to C7 linear hydrocarbyl moiety, optionallysubstituted with hydroxyl groups, and/or optionally comprising one ormore ether linkages.

In other embodiments, suitable carboxylic acid ester substrates may alsoinclude acetylated saccharides selected from the group consisting ofacetylated mono-, di-, and polysaccharides. In preferred embodiments,the acetylated saccharides include acetylated mono-, di-, andpolysaccharides. In other embodiments, the acetylated saccharides areselected from the group consisting of acetylated xylan, fragments ofacetylated xylan, acetylated xylose (such as xylose tetraacetate),acetylated glucose (such as glucose pentaacetate),β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal,tri-O-acetyl-D-glucal, and acetylated cellulose. In preferredembodiments, the acetylated saccharide is selected from the groupconsisting of β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucal, and acetylatedcellulose. As such, acetylated carbohydrates may be suitable substratesfor generating percarboxylic acids using the present methods and systems(i.e., in the presence of a peroxygen source).

In additional embodiments, the carboxylic acid ester substrate may bemonoacetin; triacetin; monopropionin; dipropionin; tripropionin;monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylosetetraacetate; acetylated xylan; acetylated xylan fragments;β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal;tri-O-acetyl-glucal; propylene glycol diacetate; ethylene glycoldiacetate; monoesters or diesters of 1,2-ethanediol; 1,2-propanediol;1,3-propanediol; 1,2-butanediol; 1,3-butanediol; 2,3-butanediol;1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol; 1,6-pentanediol;1,2-hexanediol; 2,5-hexanediol; 1,6-hexanediol; and mixtures thereof. Inpreferred embodiments of the present methods and systems, the substratecomprises triacetin.

The carboxylic acid ester is present in the reaction formulation at aconcentration sufficient to produce the desired concentration of peracidupon enzyme-catalyzed perhydrolysis. The carboxylic acid ester need notbe completely soluble in the reaction formulation, but has sufficientsolubility to permit conversion of the ester by the perhydrolasecatalyst to the corresponding peracid. The carboxylic acid ester ispresent in the reaction formulation at a concentration of 0.05 wt % to40 wt % of the reaction formulation, preferably at a concentration of0.1 wt % to 20 wt % of the reaction formulation, and more preferably ata concentration of 0.5 wt % to 10 wt % of the reaction formulation.

The peroxygen source may include, but is not limited to, hydrogenperoxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct(carbamide peroxide)) perborate salts and percarbonate salts. Theconcentration of peroxygen compound in the reaction formulation mayrange from 0.0033 wt % to about 50 wt %, preferably from 0.033 wt % toabout 40 wt %, more preferably from 0.33 wt % to about 30 wt %.

Many perhydrolase catalysts (whole cells, permeabilized whole cells, andpartially purified whole cell extracts) have been reported to havecatalase activity (EC 1.11.1.6). Catalases catalyze the conversion ofhydrogen peroxide into oxygen and water. In one aspect, theperhydrolysis catalyst lacks catalase activity. In another aspect, acatalase inhibitor is added to the reaction formulation. Examples ofcatalase inhibitors include, but are not limited to, sodium azide andhydroxylamine sulfate. One of skill in the art can adjust theconcentration of catalase inhibitor as needed. The concentration of thecatalase inhibitor typically ranges from 0.1 mM to about 1 M; preferablyabout 1 mM to about 50 mM; more preferably from about 1 mM to about 20mM. In one aspect, sodium azide concentration typically ranges fromabout 20 mM to about 60 mM while hydroxylamine sulfate is concentrationis typically about 0.5 mM to about 30 mM, preferably about 10 mM.

In another embodiment, the enzyme catalyst lacks significant catalaseactivity or is engineered to decrease or eliminate catalase activity.The catalase activity in a host cell can be down-regulated or eliminatedby disrupting expression of the gene(s) responsible for the catalaseactivity using well known techniques including, but not limited to,transposon mutagenesis, RNA antisense expression, targeted mutagenesis,and random mutagenesis. In a preferred embodiment, the gene(s) encodingthe endogenous catalase activity are down-regulated or disrupted (i.e.,knocked-out). As used herein, a “disrupted” gene is one where theactivity and/or function of the protein encoded by the modified gene isno longer present. Means to disrupt a gene are well-known in the art andmay include, but are not limited to, insertions, deletions, or mutationsto the gene so long as the activity and/or function of the correspondingprotein is no longer present. In a further preferred embodiment, theproduction host is an E. coli production host comprising a disruptedcatalase gene selected from the group consisting of katG and katE (seePublished U.S. Patent Application No. 2008-0176299). In anotherembodiment, the production host is an E. coli strain comprising adown-regulation and/or disruption in both katgl and a katE catalasegenes.

The concentration of the catalyst in the aqueous reaction formulationdepends on the specific catalytic activity of the catalyst, and ischosen to obtain the desired rate of reaction. The weight of catalyst inperhydrolysis reactions typically ranges from 0.0001 mg to 10 mg per mLof total reaction volume, preferably from 0.001 mg to 2.0 mg per mL. Thecatalyst may also be immobilized on a soluble or insoluble support usingmethods well-known to those skilled in the art; see for example,Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997. The use of immobilized catalystspermits the recovery and reuse of the catalyst in subsequent reactions.The enzyme catalyst may be in the form of whole microbial cells,permeabilized microbial cells, microbial cell extracts,partially-purified or purified enzymes, and mixtures thereof.

In one aspect, the concentration of peracid generated by the combinationof chemical perhydrolysis and enzymatic perhydrolysis of the carboxylicacid ester is sufficient to provide an effective concentration ofperacid for bleaching or disinfection at a desired pH. In anotheraspect, the present methods provide combinations of enzymes and enzymesubstrates to produce the desired effective concentration of peracid,where, in the absence of added enzyme, there is a significantly lowerconcentration of peracid produced. Although there may in some cases besubstantial chemical perhydrolysis of the enzyme substrate by directchemical reaction of inorganic peroxide with the enzyme substrate, theremay not be a sufficient concentration of peracid generated to provide aneffective concentration of peracid in the desired applications, and asignificant increase in total peracid concentration is achieved by theaddition of an appropriate perhydrolase catalyst to the reactionformulation.

The concentration of peracid generated (such as peracetic acid) by theperhydrolysis of at least one carboxylic acid ester is at least about 20ppm, preferably at least 100 ppm, more preferably at least about 200 ppmperacid, more preferably at least 300 ppm, more preferably at least 500ppm, more preferably at least 700 ppm, more preferably at least about1000 ppm peracid, most preferably at least 2000 ppm peracid within 10minutes, preferably within 5 minutes, more preferably within 1 minute ofinitiating the perhydrolysis reaction. The product formulationcomprising the peracid may be optionally diluted with water, or asolution predominantly comprised of water, to produce a formulation withthe desired lower concentration of peracid. In one aspect, the reactiontime required to produce the desired concentration of peracid is notgreater than about two hours, preferably not greater than about 30minutes, more preferably not greater than about 10 minutes, and mostpreferably in about 5 minutes or less. In other aspects, a hard surfaceor inanimate object contaminated with a biological contaminant(s) iscontacted with the peracid formed in accordance with the processesdescribed herein within about 5 minutes to about 168 hours of combiningsaid reaction components, or within about 5 minutes to about 48 hours,or within about 5 minutes to 2 hours of combining said reactioncomponents, or any such time interval therein.

In another aspect, the peroxycarboxylic acid formed in accordance withthe processes describe herein is used in a laundry care applicationwherein the peroxycarboxylic acid is contacted with an article ofclothing or a textile to provide a benefit, such as disinfecting,bleaching, destaining, sanitizing, deodorizing or a combination thereof.The peroxycarboxylic acid may be used in a variety of laundry careproducts including, but not limited to, textile pre-wash treatments,laundry detergents, stain removers, bleaching compositions, deodorizingcompositions, and rinsing agents. In one embodiment, the present processto produce a peroxycarboxylic acid for a target surface is conducted insitu.

In the context of laundry care applications, the term “contacting anarticle of clothing or textile” means that the article of clothing ortextile is exposed to a formulation disclosed herein. To this end, thereare a number of formats the formulation may be used to treat articles ofclothing or textiles including, but not limited to, liquid, solids, gel,paste, bars, tablets, spray, foam, powder, or granules and can bedelivered via hand dosing, unit dosing, dosing from a substrate,spraying and automatic dosing from a laundry washing or drying machine.Granular compositions can also be in compact form; liquid compositionscan also be in a concentrated form.

When the formulations disclosed herein are used in a laundry machine,the formulation can further contain components typical to laundrydetergents. For example, typical components included, but are notlimited to, surfactants, bleaching agents, bleach activators, additionalenzymes, suds suppressors, dispersants, lime-soap dispersants, soilsuspension and anti-redeposition agents, softening agents, corrosioninhibitors, tarnish inhibitors, germicides, pH adjusting agents,non-builder alkalinity sources, chelating agents, organic and/orinorganic fillers, solvents, hydrotropes, optical brighteners, dyes, andperfumes.

The formulations disclosed herein can also be used as detergent additiveproducts in solid or liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

In connection with the present systems and methods for laundry carewhere the peracid is generated for one or more of bleaching, stainremoval, and odor reduction, the concentration of peracid generated(e.g., peracetic acid) by the perhydrolysis of at least one carboxylicacid ester may be at least about 2 ppm, preferably at least 20 ppm,preferably at least 100 ppm, and more preferably at least about 200 ppmperacid. In connection with the present systems and methods for laundrycare where the peracid is generated for disinfection or sanitization,the concentration of peracid generated (e.g., peracetic acid) by theperhydrolysis of at least one carboxylic acid ester may be at leastabout 2 ppm, more preferably at least 20 ppm, more preferably at least200 ppm, more preferably at least 500 ppm, more preferably at least 700ppm, more preferably at least about 1000 ppm peracid, most preferably atleast 2000 ppm peracid within 10 minutes, preferably within 5 minutes,and most preferably within 1 minute of initiating the perhydrolysisreaction. The product mixture comprising the peracid may be optionallydiluted with water, or a solution predominantly comprised of water, toproduce a mixture with the desired lower concentration of peracid. Inone aspect of the present methods and systems, the reaction timerequired to produce the desired concentration of peracid is not greaterthan about two hours, preferably not greater than about 30 minutes, morepreferably not greater than about 10 minutes, even more preferably notgreater than about 5 minutes, and most preferably in about 1 minute orless.

The temperature of the reaction is chosen to control both the reactionrate and the stability of the enzyme catalyst activity. The temperatureof the reaction may range from just above the freezing point of thereaction formulation (approximately 0° C.) to about 95° C., with apreferred range of reaction temperature of from about 5° C. to about 55°C.

The pH of the final reaction formulation containing peracid is fromabout 2 to about 9, preferably from about 3 to about 8, more preferablyfrom about 5 to about 8, even more preferably about 5.5 to about 8, andyet even more preferably about 6.0 to about 7.5. In another embodiment,the pH of the reaction formulation is acidic (pH<7). The pH of thereaction, and of the final reaction formulation, may optionally becontrolled by the addition of a suitable buffer, including, but notlimited to, bicarbonate, pyrophosphate, phosphate, methylphosphonate,citrate, acetate, malate, fumarate, tartrate maleate or succinate. Theconcentration of buffer, when employed, is typically from 0.1 mM to 1.0M, preferably from 1 mM to 300 mM, most preferably from 10 mM to 100 mM.

In another aspect, the enzymatic perhydrolysis reaction formulation maycontain an organic solvent that acts as a dispersant to enhance the rateof dissolution of the carboxylic acid ester in the reaction formulation.Such solvents include, but are not limited to, propylene glycol methylether, acetone, cyclohexanone, diethylene glycol butyl ether,tripropylene glycol methyl ether, diethylene glycol methyl ether,propylene glycol butyl ether, dipropylene glycol methyl ether,cyclohexanol, benzyl alcohol, isopropanol, ethanol, propylene glycol,and mixtures thereof.

In another aspect, the enzymatic perhydrolysis product may containadditional components that provide desirable functionality. Theseadditional components include, but are not limited to, buffers,detergent builders, thickening agents, emulsifiers, surfactants, wettingagents, corrosion inhibitors (such as benzotriazole), enzymestabilizers, and peroxide stabilizers (e.g., metal ion chelatingagents). Many of the additional components are well known in thedetergent industry (see, for example, U.S. Pat. No. 5,932,532; herebyincorporated by reference). Examples of emulsifiers include, but are notlimited to, polyvinyl alcohol or polyvinylpyrrolidone. Examples ofthickening agents include, but are not limited to LAPONITE® RD, cornstarch, PVP, CARBOWAX®, CARBOPOL®, CABOSIL®, polysorbate 20, PVA, andlecithin. Examples of buffering systems include, but are not limited to,sodium phosphate monobasic/sodium phosphate dibasic; sulfamicacid/triethanolamine; citric acid/triethanolamine; tartaricacid/triethanolamine; succinic acid/triethanolamine; and aceticacid/triethanolamine. Examples of surfactants include, but are notlimited to, a) non-ionic surfactants such as block copolymers ofethylene oxide or propylene oxide, ethoxylated or propoxylated linearand branched primary and secondary alcohols, and aliphatic phosphineoxides; b) cationic surfactants such as quaternary ammonium compounds,particularly quaternary ammonium compounds having a C8-C20 alkyl groupbound to a nitrogen atom additionally bound to three C1-C2 alkyl groups;c) anionic surfactants such as alkane carboxylic acids (e.g., C8-C20fatty acids), alkyl phosphonates, alkane sulfonates (e.g., sodiumdodecylsulphate “SDS”) or linear or branched alkyl benzene sulfonates,alkene sulfonates; and d) amphoteric and zwitterionic surfactants, suchas aminocarboxylic acids, aminodicarboxylic acids, alkybetaines, andmixtures thereof. Additional components may include fragrances, dyes,stabilizers of hydrogen peroxide (e.g., metal chelators such as1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST® 2010, Solutia Inc.,St. Louis, Mo. and ethylenediaminetetraacetic acid (EDTA)), TURPINAL® SL(CAS#2809-21-4), DEQUEST® 0520, DEQUEST® 0531, stabilizers of enzymeactivity (e.g., polyethylene glycol (PEG)), and detergent builders.

In Situ Production of Peracids using a Perhydrolase Catalyst

Cephalosporin C deacetylases (E.C. 3.1.1.41; systematic namecephalosporin C acetylhydrolases; CAHs) are enzymes having the abilityto hydrolyze the acetyl ester bond on cephalosporins such ascephalosporin C, 7-aminocephalosporanic acid, and7-(thiophene-2-acetamido)cephalosporanic acid (Abbott, B. and Fukuda,D., Appl. Microbiol. 30(3):413-419 (1975)). CAHs belong to a largerfamily of structurally related enzymes referred to as the carbohydrateesterase family seven (“CE-7”; Coutinho, P. M., Henrissat, B., supra).

The CE-7 carbohydrate esterase family includes both CAHs and acetylxylan esterases (AXEs; E.C. 3.1.1.72). CE-7 family members share acommon structural motif and are quite unusual in that they typicallyexhibit ester hydrolysis activity for both acetylatedxylooligosaccharides and acetylated cephalosporin C, suggesting that theCE-7 family represents a single class of proteins with a multifunctionaldeacetylase activity against a range of small substrates (Vincent etal., supra). Vincent et al. describes the structural similarity amongthe members of this family and defines a signature sequence motifcharacteristic of the CE-7 family.

Members of the CE-7 family are found in plants, fungi (e.g.,Cephalosporidium acremonium), yeasts (e.g., Rhodosporidium toruloides,Rhodotorula glutinis), and bacteria such as Thermoanaerobacterium sp.;Norcardia lactamdurans, and various members of the genus Bacillus(Politino et al., Appl. Environ. Microbiol., 63(12):4807-4811 (1997);Sakai et al., J. Ferment. Bioeng. 85:53-57 (1998); Lorenz, W. andWiegel, J., J. Bacteriol 179:5436-5441 (1997); Cardoza et al., Appl.Microbiol. Biotechnol., 54(3):406-412 (2000); Mitsushima et al., supra;Abbott, B. and Fukuda, D., Appl. Microbiol. 30(3):413-419 (1975);Vincent et al., supra; Takami et al., NAR, 28(21):4317-4331 (2000); Reyet al., Genome Biol., 5(10): article 77 (2004); Degrassi et al.,Microbiology., 146:1585-1591 (2000); U.S. Pat. No. 6,645,233;. U.S. Pat.No. 5,281,525; U.S. Pat. No. 5,338,676; and WO 99/03984.

WO2007/070609 and U.S. Patent Application Publication Nos. 2008/0176299and 2008/176783 to DiCosimo et al. disclose various enzymes structurallyclassified as CE-7 enzymes that have perhydrolysis activity suitable forproducing efficacious concentrations of peracids from a variety ofcarboxylic acid ester substrates when combined with a source ofperoxygen. Variant CE-7 enzymes having improved perhydrolysis activityare also described in co-owned U.S. Pat. No. 8,062,875 (incorporatedherein by reference in its entirety).

The present method produces industrially-useful, efficaciousconcentrations of peracids in situ under aqueous reaction conditionsusing the perhydrolase activity of an enzyme belonging to the CE-7family of carbohydrate esterases.

HPLC Assay Method for Determining the Concentration of Peracid andHydrogen Peroxide.

A variety of analytical methods can be used in the present methods toanalyze the reactants and products including, but not limited to,titration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), capillary electrophoresis(CE), the analytical procedure described by U. Karst et al., (Anal.Chem., 69(17):3623-3627 (1997)), and the2,2′-azino-bis(3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S.Minning, et al., Analytica Chimica Acta 378:293-298 (1999) and WO2004/058961 A1) as described in the present examples.

Determination of Minimum Biocidal Concentration of Peracids

The method described by J. Gabrielson, et al. (J. Microbiol. Methods 50:63-73 (2002)) can be employed for determination of the Minimum BiocidalConcentration (MBC) of peracids, or of hydrogen peroxide and enzymesubstrates. The assay method is based on XTT reduction inhibition, whereXTT((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to, viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Uses of Enzymatically Prepared Peroxycarboxylic acid Compositions

The enzyme catalyst-generated peroxycarboxylic acid produced accordingto the present method can be used in a variety of hard surface/inanimateobject applications for reduction of concentrations of biologicalcontaminants, such as decontamination of medical instruments (e.g.,endoscopes), textiles (e.g., garments, carpets), food preparationsurfaces, food storage and food-packaging equipment, materials used forthe packaging of food products, chicken hatcheries and grow-outfacilities, animal enclosures, and spent process waters that havemicrobial and/or virucidal activity. The enzyme-generatedperoxycarboxylic acids may be used in formulations designed toinactivate prions (e.g., certain proteases) to additionally providebiocidal activity. In a preferred aspect, the present peroxycarboxylicacid compositions are particularly useful as a disinfecting agent fornon-autoclavable medical instruments and food packaging equipment. Asthe peroxycarboxylic acid-containing formulation may be prepared usingGRAS or food-grade components (enzyme, enzyme substrate, hydrogenperoxide, and buffer), the enzyme-generated peroxycarboxylic acid mayalso be used for decontamination of animal carcasses, meat, fruits andvegetables, or for decontamination of prepared foods. Theenzyme-generated peroxycarboxylic acid may be incorporated into aproduct whose final form is a powder, liquid, gel, film, solid oraerosol. The enzyme-generated peroxycarboxylic acid may be diluted to aconcentration that still provides an efficacious decontamination.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can be used to disinfect surfaces and/or objectscontaminated (or suspected of being contaminated) with biologicalcontaminants by contacting the surface or object with the productsproduced by the present processes. As used herein, “contacting” refersto placing a disinfecting composition comprising an effectiveconcentration of peroxycarboxylic acid in contact with the surface orinanimate object suspected of contamination with a biologicalcontaminant for a period of time sufficient to clean and disinfect.Contacting includes spraying, treating, immersing, flushing, pouring onor in, mixing, combining, painting, coating, applying, affixing to andotherwise communicating a peroxycarboxylic acid solution or compositioncomprising an efficacious concentration of peroxycarboxylic acid, or asolution or composition that forms an efficacious concentration ofperoxycarboxylic acid, with the surface or inanimate object suspected ofbeing contaminated with a concentration of a biological contaminant. Thedisinfectant compositions may be combined with a cleaning composition toprovide both cleaning and disinfection. Alternatively, a cleaning agent(e.g., a surfactant or detergent) may be incorporated into theformulation to provide both cleaning and disinfection in a singlecomposition.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can also contain at least one additionalantimicrobial agent, combinations of prion-degrading proteases, avirucide, a sporicide, or a biocide. Combinations of these agents withthe peroxycarboxylic acid produced by the claimed processes can providefor increased and/or synergistic effects when used to clean anddisinfect surfaces and/or objects contaminated (or suspected of beingcontaminated) with biological contaminants. Suitable antimicrobialagents include carboxylic esters (e.g., p-hydroxy alkyl benzoates andalkyl cinnamates); sulfonic acids (e.g., dodecylbenzene sulfonic acid);iodo-compounds or active halogen compounds (e.g., elemental halogens,halogen oxides (e.g., NaOCl, HOCl, HOBr, ClO₂), iodine, interhalides(e.g., iodine monochloride, iodine dichloride, iodine trichloride,iodine tetrachloride, bromine chloride, iodine monobromide, or iodinedibromide), polyhalides, hypochlorite salts, hypochlorous acid,hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins,chlorine dioxide, and sodium chlorite); organic peroxides includingbenzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygengenerators, and mixtures thereof; phenolic derivatives (such as o-phenylphenol, o-benzyl-p-chlorophenol, tert-amyl phenol and C₁-C₆ alkylhydroxy benzoates); quaternary ammonium compounds (such asalkyldimethylbenzyl ammonium chloride, dialkyldimethyl ammonium chlorideand mixtures thereof); and mixtures of such antimicrobial agents, in anamount sufficient to provide the desired degree of microbial protection.Effective amounts of antimicrobial agents include about 0.001 wt % toabout 60 wt % antimicrobial agent, about 0.01 wt % to about 15 wt %antimicrobial agent, or about 0.08 wt % to about 2.5 wt % antimicrobialagent.

In one aspect, the peroxycarboxylic acids formed by the present processcan be used to reduce the concentration of viable biologicalcontaminants (such as a viable microbial population) when applied onand/or at a locus. As used herein, a “locus” comprises part or all of atarget surface suitable for disinfecting or bleaching. Target surfacesinclude all surfaces that can potentially be contaminated withbiological contaminants. Non-limiting examples include equipmentsurfaces found in the food or beverage industry (such as tanks,conveyors, floors, drains, coolers, freezers, equipment surfaces, walls,valves, belts, pipes, drains, joints, crevasses, combinations thereof,and the like); building surfaces (such as walls, floors and windows);non-food-industry related pipes and drains, including water treatmentfacilities, pools and spas, and fermentation tanks; hospital orveterinary surfaces (such as walls, floors, beds, equipment (such asendoscopes), clothing worn in hospital/veterinary or other healthcaresettings, including clothing, scrubs, shoes, and other hospital orveterinary surfaces); restaurant surfaces; bathroom surfaces; toilets;clothes and shoes; surfaces of barns or stables for livestock, such aspoultry, cattle, dairy cows, goats, horses and pigs; hatcheries forpoultry or for shrimp; and pharmaceutical or biopharmaceutical surfaces(e.g., pharmaceutical or biopharmaceutical manufacturing equipment,pharmaceutical or biopharmaceutical ingredients, pharmaceutical orbiopharmaceutical excipients). Additional hard surfaces also includefood products, such as beef, poultry, pork, vegetables, fruits, seafood,combinations thereof, and the like. The locus can also include waterabsorbent materials such as infected linens or other textiles. The locusalso includes harvested plants or plant products including seeds, corms,tubers, fruit, and vegetables, growing plants, and especially cropgrowing plants, including cereals, leaf vegetables and salad crops, rootvegetables, legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, vinyl, linoleum, and carpet.

The peroxycarboxylic acids formed by the present process may be used toprovide a benefit to a textile including, but not limited to, bleaching,disinfecting, sanitizing, destaining, and deodorizing. Theperoxycarboxylic acids formed by the present process may be used in anynumber of laundry care products including, but not limited to, textilepre-wash treatments, laundry detergents, stain removers, bleachingcompositions, deodorizing compositions, and rinsing agents.

Recombinant Microbial Expression

The genes and gene products of the instant sequences may be produced inheterologous host cells, particularly in the cells of microbial hosts.Preferred heterologous host cells for expression of the instant genesand nucleic acid molecules are microbial hosts that can be found withinthe fungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi maysuitably host the expression of the present nucleic acid molecules. Theperhydrolase may be expressed intracellularly, extracellularly, or acombination of both intracellularly and extracellularly, whereextracellular expression renders recovery of the desired protein from afermentation product more facile than methods for recovery of proteinproduced by intracellular expression. Transcription, translation and theprotein biosynthetic apparatus remain invariant relative to the cellularfeedstock used to generate cellular biomass; functional genes will beexpressed regardless. Examples of host strains include, but are notlimited to, bacterial, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida,Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas,Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium,Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium,Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia,Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylomicrobium, Methylocystis,Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus,Methanobacterium, Klebsiella, and Myxococcus. In one embodiment,bacterial host strains include Escherichia, Bacillus, Kluyveromyces, andPseudomonas. In a preferred embodiment, the bacterial host cell isEscherichia coli.

Large-scale microbial growth and functional gene expression may use awide range of simple or complex carbohydrates, organic acids andalcohols or saturated hydrocarbons, such as methane or carbon dioxide inthe case of photosynthetic or chemoautotrophic hosts, the form andamount of nitrogen, phosphorous, sulfur, oxygen, carbon or any tracemicronutrient including small inorganic ions. The regulation of growthrate may be affected by the addition, or not, of specific regulatorymolecules to the culture and which are not typically considered nutrientor energy sources.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell and/or native to theproduction host, although such control regions need not be so derived.

Initiation control regions or promoters, which are useful to driveexpression of the present cephalosporin C deacetylase coding region inthe desired host cell are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving these genes is suitablefor the present invention including, but not limited to, CYC1, HIS3,GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI(useful for expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, araB, tet, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in Escherichia coli) as well as the amy, apr, nprpromoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred host cell. In one embodiment, the inclusion of atermination control region is optional. In another embodiment, thechimeric gene includes a termination control region derived from thepreferred host cell.

Industrial Production

A variety of culture methodologies may be applied to produce theperhydrolase catalyst. For example, large-scale production of a specificgene product overexpressed from a recombinant microbial host may beproduced by batch, fed-batch, and continuous culture methodologies.Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, Second Edition, Sinauer Associates,Inc., Sunderland, Mass. (1989) and Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992).

Commercial production of the desired perhydrolase catalyst may also beaccomplished with a continuous culture. Continuous cultures are an opensystem where a defined culture media is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Recovery of the desired perhydrolase catalysts from a batchfermentation, fed-batch fermentation, or continuous culture, may beaccomplished by any of the methods that are known to those skilled inthe art. For example, when the enzyme catalyst is producedintracellularly, the cell paste is separated from the culture medium bycentrifugation or membrane filtration, optionally washed with water oran aqueous buffer at a desired pH, then a suspension of the cell pastein an aqueous buffer at a desired pH is homogenized to produce a cellextract containing the desired enzyme catalyst. The cell extract mayoptionally be filtered through an appropriate filter aid such as celiteor silica to remove cell debris prior to a heat-treatment step toprecipitate undesired protein from the enzyme catalyst solution. Thesolution containing the desired enzyme catalyst may then be separatedfrom the precipitated cell debris and protein by membrane filtration orcentrifugation, and the resulting partially-purified enzyme catalystsolution concentrated by additional membrane filtration, then optionallymixed with an appropriate carrier (for example, maltodextrin, phosphatebuffer, citrate buffer, or mixtures thereof) and spray-dried to producea solid powder comprising the desired enzyme catalyst.

When an amount, concentration, or other value or parameter is giveneither as a range, preferred range, or a list of upper preferable valuesand lower preferable values, this is to be understood as specificallydisclosing all ranges formed from any pair of any upper range limit orpreferred value and any lower range limit or preferred value, regardlessof whether ranges are separately disclosed. Where a range of numericalvalues is recited herein, unless otherwise stated, the range is intendedto include the endpoints thereof, and all integers and fractions withinthe range. It is not intended that the scope be limited to the specificvalues recited when defining a range.

General Methods

The following examples are provided to demonstrate preferredembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the methods disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the presently disclosed methods.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.) orSigma-Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “4”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “ppm”means part(s) per million, “wt” means weight, “wt %” means weightpercent, “g” means gram(s), “mg” means milligram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “g” means gravity, “HPLC” meanshigh performance liquid chromatography, “dd H₂O” means distilled anddeionized water, “dcw” means dry cell weight, “ATCC” or “ATCC®” meansthe American Type Culture Collection (Manassas, Va.), “U” means unit(s)of perhydrolase activity, “rpm” means revolution(s) per minute, “Tg”means glass transition temperature, and “EDTA” meansethylenediaminetetraacetic acid.

Example 1 Construction of a katG Catalase Disrupted E. coli Strain

The coding region of the kanamycin resistance gene (kan; SEQ ID NO:26)was amplified from the plasmid pKD13 (SEQ ID NO:27) by PCR (0.5 min at94° C., 0.5 min at 55° C., 1 min at 70° C., 30 cycles) using primersidentified as SEQ ID NO:28 and SEQ ID NO:29 to generate the PCR productidentified as SEQ ID NO:30. The katG nucleic acid sequence is providedas SEQ ID NO:31 and the corresponding amino acid sequence is SEQ IDNO:32. E. coli MG1655 (ATCC® 47076™) was transformed with thetemperature-sensitive plasmid pKD46 (SEQ ID NO:33), which contains theλ-Red recombinase genes (Datsenko and Wanner, (2000), PNAS USA97:6640-6645), and selected on LB-amp plates for 24 h at 30° C.MG1655/pKD46 was transformed with 50-500 ng of the PCR product byelectroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W, 25pF), and selected on LB-kan plates for 24 h at 37° C. Several colonieswere streaked onto LB-kan plates and incubated overnight at 42° C. tocure the pKD46 plasmid. Colonies were checked to confirm a phenotype ofkanR/ampS. Genomic DNA was isolated from several colonies using thePUREGENE® DNA purification system (Gentra Systems, Minneapolis, Minn.),and checked by PCR to confirm disruption of the katG gene using primersidentified as SEQ ID NO:34 and SEQ ID NO:35. Several katG-disruptedstrains were transformed with the temperature-sensitive plasmid pCP20(SEQ ID NO:36), which contains the FLP recombinase, used to excise thekan gene, and selected on LB-amp plates for 24 h at 37° C. Severalcolonies were streaked onto LB plates and incubated overnight at 42° C.to cure the pCP20 plasmid. Two colonies were checked to confirm aphenotype of kanS/ampS, and called MG1655 KatG1 and MG1655 KatG2.

Example 2 Construction of a katE Catalase Disrupted E. coli Strain

The kanamycin resistance gene (SEQ ID NO:26) was amplified from theplasmid pKD13 (SEQ ID NO:27) by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:37and SEQ ID NO:38 to generate the PCR product identified as SEQ ID NO:39.The katE nucleic acid sequence is provided as SEQ ID NO:40 and thecorresponding amino acid sequence is SEQ ID NO:41. E. coli MG1655 (ATCC®47076™) was transformed with the temperature-sensitive plasmid pKD46(SEQ ID NO:33), which contains the λ-Red recombinase genes, and selectedon LB-amp plates for 24 h at 30° C. MG1655/pKD46 was transformed with50-500 ng of the PCR product by electroporation (BioRad Gene Pulser, 0.2cm cuvette, 2.5 kV, 200 W, 25 μF), and selected on LB-kan plates for 24h at 37° C. Several colonies were streaked onto LB-kan plates andincubated overnight at 42° C. to cure the pKD46 plasmid. Colonies werechecked to confirm a phenotype of kanR/ampS. Genomic DNA was isolatedfrom several colonies using the PUREGENE® DNA purification system, andchecked by PCR to confirm disruption of the katE gene using primersidentified as SEQ ID NO:42 and SEQ ID NO:43. Several katE-disruptedstrains were transformed with the temperature-sensitive plasmid pCP20(SEQ ID NO:36), which contains the FLP recombinase, used to excise thekan gene, and selected on LB-amp plates for 24 h at 37° C. Severalcolonies were streaked onto LB plates and incubated overnight at 42° C.to cure the pCP20 plasmid. Two colonies were checked to confirm aphenotype of kanS/ampS, and called MG1655 KatE1 and MG1655 KatE2.

Example 3 Construction of a katG Catalase and katE Catalase Disrupted E.coli Strain (KLP18)

The Kanamycin Resistance Gene (Seq Id No:26) was Amplified from theplasmid pKD13 (SEQ ID NO:27) by PCR (0.5 min at 94° C., 0.5 min at 55°C., 1 min at 70° C., 30 cycles) using primers identified as SEQ ID NO:37and SEQ ID NO:38 to generate the PCR product identified as SEQ ID NO:39.E. coli MG1655 KatG1 (EXAMPLE 1) was transformed with thetemperature-sensitive plasmid pKD46 (SEQ ID NO:33), which contains theλ-Red recombinase genes, and selected on LB-amp plates for 24 h at 30°C. MG1655 KatG1/pKD46 was transformed with 50-500 ng of the PCR productby electroporation (BioRad Gene Pulser, 0.2 cm cuvette, 2.5 kV, 200 W,25 pF), and selected on LB-kan plates for 24 h at 37° C. Severalcolonies were streaked onto LB-kan plates and incubated overnight at 42°C. to cure the pKD46 plasmid. Colonies were checked to confirm aphenotype of kanR/ampS. Genomic DNA was isolated from several coloniesusing the PUREGENE® DNA purification system, and checked by PCR toconfirm disruption of the katE gene using primers identified as SEQ IDNO:42 and SEQ ID NO:43. Several katE-disrupted strains (Δ katE) weretransformed with the temperature-sensitive plasmid pCP20 (SEQ ID NO:36),which contains the FLP recombinase, used to excise the kan gene, andselected on LB-amp plates for 24 h at 37° C. Several colonies werestreaked onto LB plates and incubated overnight at 42° C. to cure thepCP20 plasmid. Two colonies were checked to confirm a phenotype ofkanS/ampS, and called MG1655 KatG1KatE18.1 and MG1655 KatG1KatE23.MG1655 KatG1KatE18.1 is designated E. coli KLP18.

Example 4 Cloning and Expression of Perhydrolase from Thermotoganeapolitana

The coding region of the gene encoding acetyl xylan esterase fromThermotoga neapolitana as reported in GENBANK® (accession numberAE000512; region 80481-81458; SEQ ID NO:44) was synthesized using codonsoptimized for expression in E. coli (DNA 2.0, Menlo Park, Calif.). Thecoding region of the gene was subsequently amplified by PCR (0.5 min at94° C., 0.5 min at 55° C., 1 min at 70° C., 30 cycles) using primersidentified as SEQ ID NO:45 and SEQ ID NO:46. The resulting nucleic acidproduct (SEQ ID NO:47) was subcloned into pTrcHis2-TOPO® to generate theplasmid identified as pSW196. The plasmid pSW196 was used to transformE. coli KLP18 (EXAMPLE 3) to generate the strain KLP18/pSW196.KLP18/pSW196 was grown in LB media at 37° C. with shaking up toOD_(600nm)=0.4-0.5, at which time IPTG was added to a finalconcentration of 1 mM, and incubation continued for 2-3 h. Cells wereharvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase at 20-40% of total soluble protein.

Example 5 Cloning and Expression of Perhydrolase from Thermotogamaritima MSB8

The coding region of the gene encoding acetyl xylan esterase fromThermotoga maritima MSB8 as reported in GENBANK® (accession#NP_(—)227893.1; SEQ ID NO:48) was synthesized (DNA 2.0, Menlo Park,Calif.). The coding region of the gene was subsequently amplified by PCR(0.5 min @ 94° C., 0.5 min @ 55° C., 1 min @ 70° C., 30 cycles) usingprimers identified as SEQ ID NO:49 and SEQ ID NO:50. The resultingnucleic acid product (SEQ ID NO:51) was cut with restriction enzymesPstI and XbaI and subcloned between the PstI and XbaI sites in pUC19 togenerate the plasmid identified as pSW207. The plasmid pSW207 was usedto transform E. coli KLP18 (EXAMPLE 3) to generate the strain identifiedas KLP18/pSW207. KLP18/pSW207 was grown in LB media at 37° C. withshaking up to OD_(600nm)=0.4-0.5, at which time IPTG was added to afinal concentration of 1 mM, and incubation continued for 2-3 h. Cellswere harvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase enzyme at 20-40% of total solubleprotein.

Example 6 Fermentation of E. coli KLP18 Transformants ExpressingPerhydrolase

A fermentor seed culture was prepared by charging a 2-L shake flask with0.5 L seed medium containing yeast extract (Amberex 695, 5.0 g/L),K₂HPO₄ (10.0 g/L), KH₂PO₄ (7.0 g/L), sodium citrate dihydrate (1.0 g/L),(NH₄)₂SO₄ (4.0 g/L), MgSO₄ heptahydrate (1.0 g/L) and ferric ammoniumcitrate (0.10 g/L). The pH of the medium was adjusted to 6.8 and themedium was sterilized in the flask. Post sterilization additionsincluded glucose (50 wt %, 10.0 mL) and 1 mL ampicillin (25 mg/mL) stocksolution. The seed medium was inoculated with a 1-mL culture of E. coliKLP18/pSW196 or E. coli KLP18/pSW207 in 20% glycerol, and cultivated at35° C. and 300 rpm. The seed culture was transferred at ca. 1-2OD_(550nm) to a 14-L fermentor (Braun Biotech, Allentown, Pa.) with 8 Lof medium at 35° C. containing KH₂PO₄ (3.50 g/L), FeSO₄ heptahydrate(0.05 g/L), MgSO₄ heptahydrate (2.0 g/L), sodium citrate dihydrate (1.90g/L), yeast extract (Amberex 695, 5.0 g/L), Biospumex153K antifoam (0.25mL/L, Cognis Corporation, Monheim, Germany), NaCl (1.0 g/L), CaCl₂dihydrate (10 g/L), and NIT trace elements solution (10 mL/L). The traceelements solution contained citric acid monohydrate (10 g/L), MnSO₄hydrate (2 g/L), NaCl (2 g/L), FeSO₄ heptahydrate (0.5 g/L), ZnSO₄heptahydrate (0.2 g/L), CuSO₄ pentahydrate (0.02 g/L) and NaMoO₄dihydrate (0.02 g/L). Post sterilization additions included glucosesolution (50% w/w, 80.0 g) and ampicillin (25 mg/mL) stock solution(16.00 mL). Glucose solution (50% w/w) was used for fed batch. Glucosefeed was initiated when glucose concentration decreased to 0.5 g/L,starting at 0.31 g feed/min and increasing progressively each hour to0.36, 0.42, 0.49, 0.57, 0.66, 0.77, 0.90, 1.04, 1.21, 1.41, and 1.63g/min respectively; the rate remained constant afterwards. Glucoseconcentration in the medium was monitored and if the concentrationexceeded 0.1 g/L the feed rate was decreased or stopped temporarily.Induction was initiated between OD_(550nm)=56 and OD_(550nm)=80 withaddition of 16 mL IPTG (0.5 M) for the various strains. The dissolvedoxygen (DO) concentration was controlled at 25% of air saturation. TheDO was controlled first by impeller agitation rate (400 to 1400 rpm) andlater by aeration rate (2 to 10 slpm). The pH was controlled at 6.8.NH₄OH (29% w/w) and H₂SO₄ (20% w/v) were used for pH control. The headpressure was 0.5 bars. The cells were harvested by centrifugation 16 hpost IPTG addition.

Example 7 Preparation of Heat-Treated Cell Extracts of CE-7Esterases/Perhydrolases

A cell extract of an E. coli transformant expressing perhydrolase fromThermotoga neapolitana (KLP18/pSW196) or Thermotoga maritima MSB8(KLP18/pSW207) was prepared by passing a suspension of cell paste (20 wt% wet cell weight) in 0.05 M potassium phosphate buffer (pH 7.0)containing dithiothreitol (1 mM) twice through a French press having aworking pressure of 16,000 psi (−110 MPa). The crude extract was thencentrifuged at 20,000×g to remove cellular debris, producing a clarifiedcell extract that was assayed for total soluble protein (BicinchoninicAcid Kit for Protein Determination, Sigma Aldrich catalog #BCA1-KT). Theclarified Thermotoga maritima MSB8 or Thermotoga neapolitanaperhydrolase-containing extract was heated for 20 min at 75° C.,followed immediately by cooling in an ice/water bath to 5° C. Theresulting mixture was centrifuged to remove precipitated protein, andthe supernatant collected and assayed for total soluble protein asbefore. SDS-PAGE of the heat-treated supernatant indicated that theperhydrolase constituted at least ca. 90% of the total soluble proteinpresent in the supernatant.

Example 8 Temperature Stability of T. neapolitana Perhydrolase/TrehaloseSpray-Dried Enzyme Powders

A set of ten aqueous mixtures were prepared that contained varyingconcentrations of the heat-treated cell extract protein of E. coliKLP18/pSW196 (≧90% T. neapolitana perhydrolase by PAGE), trehalose(Cargill), and, optionally, polysorbate 80 (p80) as surfactant in sodiumbicarbonate buffer (50 mM, pH=8.1) (Table 1). These solutions werespray-dried using a Buchi B-290 glass-chamber spray dryer (inlettemperature=170° C., exit temperature=90° C., feed rate=3 mL/min to 10mL/min) to produce ten spray-dried enzyme powders; the weight percentprotein in the powders was determined using the BCA (Bicinchoninic acid)protein assay, and the glass transition temperatures (Tg) of thesepowders were measured using modulated differential scanning calorimetry(Table 1).

TABLE 1 Composition of protein/excipient solutions used to produce T.neapolitana perhydrolase/trehalose spray-dried enzyme powders, and Tg ofcorresponding powders. wt % protein Tg of in protein/ protein/ protein/protein/ excipient excipient trehalose protein excipient/ p80 excipientexcipient powder solution (g/L) (g/L) protein (g/L) powder powder (° C.)S1-1 52.5 35 1.5 0.25 P1-2 39.2 42 S2-1 100 50 2.0 0 P2-2 32.5 48 S3-1100 50 2.0 0.50 P3-2 33.2 40 S4-1 50 50 1.0 0 P4-2 45.1 40 S5-1 50 501.0 0.50 P5-2 46.7 54 S6-1 40 20 2.0 0 P6-2 31.4 44 S7-1 40 20 2.0 0.50P7-2 32.5 45 S8-1 20 20 1.0 0 P8-2 47.8 38 S9-1 20 20 1.0 0.50 P9-2 46.658 S10-1 52.5 35 1.5 0.25 P10-2 37.8 21

The spray-dried enzyme powders were stored in sealed vials at 40° C. andsampled at one-week intervals, and the samples assayed for theconcentration of peracetic acid produced in 5 minutes in reactionscontaining T. neapolitana perhydrolase (50 μg protein/mL), H₂O₂ (100mM), triacetin (100 mM) and TURPINAL® SL (500 ppm) in sodium bicarbonatebuffer (50 mM, pH 7.2) at 25° C., and analyzed for production ofperacetic acid using a modification of the analytical method reported byKarst et al. (below).

A sample (0.040 mL) of the reaction mixture was removed at apredetermined time (5 min) and immediately mixed with 0.960 mL of 5 mMphosphoric acid in water to terminate the reaction by adjusting the pHof the diluted sample to less than pH 4. The resulting solution wasfiltered using an ULTRAFREE® MC-filter unit (30,000 Normal MolecularWeight Limit (NMWL), Millipore Corp., Billerica, Mass.; cat #UFC3LKT 00)by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of theresulting filtrate was transferred to a 1.5-mL screw cap HPLC vial(Agilent Technologies, Palo Alto, Calif.; #5182-0715) containing 0.300mL of deionized water, then 0.100 mL of 20 mM MTS (methyl-p-tolylsulfide) in acetonitrile was added, the vial capped, and the contentsbriefly mixed prior to a 10 min incubation at ca. 25° C. in the absenceof light. To the vial was then added 0.400 mL of acetonitrile and 0.100mL of a solution of triphenylphosphine (TPP, 40 mM) in acetonitrile, thevial re-capped, and the resulting solution mixed and incubated at ca.25° C. for 30 min in the absence of light. To the vial was then added0.100 mL of 10 mM N,N-diethyl-m-toluamide (DEET; HPLC external standard)and the resulting solution analyzed by HPLC for MTSO (methyl-p-tolylsulfoxide), the stoichiometric oxidation product produced by reaction ofMTS with peracetic acid. A control reaction was run in the absence ofadded extract protein or triacetin to determine the rate of oxidation ofMTS in the assay mixture by hydrogen peroxide, for correction of therate of peracetic acid production for background MTS oxidation. HPLCmethod: Supelco Discovery C8 column (10-cm×4.0-mm, 5 μm) (cat.#569422-U) with Supelco Supelguard Discovery C8 precolumn(Sigma-Aldrich; cat #59590-U); 10 microliter injection volume; gradientmethod with CH₃CN (Sigma-Aldrich; catalog #270717) and deionized waterat 1.0 mL/min and ambient temperature.

TABLE 2 HPLC Gradient for analysis of peracetic acid. Time (min:sec) (%CH₃CN) 0:00 40 3:00 40 3:10 100 4:00 100 4:10 40 7:00 (stop) 40

The perhydrolytic activity of the T. neapolitana perhydrolase/trehalosespray-dried powder was stable over eight weeks of storage at 40° C.(Table 3).

TABLE 3 Temperature stability of T. neapolitana perhydrolase/trehalosespray- dried enzyme powders during storage at 40° C. PAA (ppm) producedin 5 min at 25° C. by reaction of triacetin (100 mM) and H₂O₂ (100 mM)in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitanaperhydrolase/ trehalose spray-dried powder (50 μg protein/mL) andTURPINAL ® SL (500 ppm). PAA (ppm) in 5 minutes time at 40° C. P1-2 P2-2P3-2 P4-2 P5-2 P6-2 P7-2 P8-2 P9-2 P10-2 initial 1855 1983 2075 20251769 1891 1902 1777 1880 1945 week 1 1872 2019 2060 1785 1776 1887 20131903 2046 2204 week 2 1830 1899 1870 1771 1833 1930 1987 1933 2146 2222week 3 1888 1974 1887 1973 1977 2223 2102 1924 2080 2104 week 4 18941878 2035 1881 1712 1918 1902 1793 1720 1988 week 5 1595 1744 1706 15651871 2052 1933 1783 1908 1985 week 6 1908 1760 1538 1545 1825 1864 17561675 1659 1758 week 7 1562 1797 1614 1487 1551 1774 1879 1927 1866 1957week 8 1881 1959 1792 1753 1939 2123 1972 1907 1902 2095

Example 9 Temperature Stability of T. neapolitana Perhydrolase/TrehaloseSpray-Dried Enzyme Powders in a Mixture of Enzyme Powder and Triacetin

The spray-dried enzyme powders prepared as described in Example 8 wereevaluated for stability when stored for eight weeks at 40° C. as amixture of the spray-dried powder in triacetin. Spray-dried enzymepowders were added to triacetin to produce a mixture containing 0.200 gof protein in 87.2 g of triacetin. The resulting mixtures were stored at40° C., and a 2.19 g sample of the well-stirred mixture was assayedweekly at 25° C. in a 100-mL reaction containing 100 mM hydrogenperoxide and TURPINAL® SL (500 ppm) in 50 mM sodium bicarbonate bufferat pH 7.2, where the resulting concentration of triacetin and proteinwas 100 mM and 50 μg/mL, respectively. Comparison of the data in Table 4with the data in Example 8, Table 3, demonstrates the instability of T.neapolitana perhydrolase/trehalose spray-dried enzyme powders whenstored as a mixture with triacetin.

TABLE 4 Temperature stability of T. neapolitana perhydrolase/trehalosespray- dried enzyme powders during storage in a mixture of enzyme powderand triacetin at 40° C. PAA (ppm) produced in 5 min at 25° C. byreaction of triacetin (100 mM) and H₂O₂ (100 mM) in sodium bicarbonatebuffer (50 mM, pH 7.2) containing T. neapolitana perhydrolase (50 μgprotein/mL) and TURPINAL ® SL (500 ppm). PAA (ppm) in 5 minutes time at40° C. P1-2 P2-2 P3-2 P4-2 P5-2 P6-2 P7-2 P8-2 P9-2 P10-2 initial 16501495 1539 1569 1666 1735 1552 1327 1712 1816 week 1 1214 1359 1597 15991589 1632 1515 1469 1421 1577 week 2 1303 1609 1580 1316 1293 1682 1353971 1402 1483 week 3 1092 1573 1568 1233 1293 1245 1268 849 1324 1388week 4 828 1563 1420 1226 1199 1608 1361 961 1172 1273 week 5 622 13401114 1294 1154 1663 1163 739 815 667 week 6 636 1301 990 970 895 1318514 313 699 372 week 7 281 998 1140 841 798 962 259 188 831 521 week 8254 569 659 563 567 483 414 323 494 321

Example 10 Temperature Stability of T. neapolitanaPerhydrolase/Maltodextrin Spray-Dried Enzyme Powder

An aqueous mixture was prepared containing heat-treated cell extractprotein of E. coli KLP18/pSW196 (34 g protein/L, ≧90% T. neapolitanaperhydrolase by PAGE) and maltodextrin (66.7 g/L MALTRIN® M100maltodextrin, 14.7 g/L MALTRIN® M250, 14.7 g/L MALTRIN® M040, GrainProcessing Corporation, Muscatine, Iowa) as excipient in 50 mM sodiumbicarbonate (pH 8.1). This solution was spray-dried using a spray dryer(GEA Niro, 3-ft diameter, inlet temperature=226° C., exittemperature=76° C., feed rate=60 g/min) to produce a spray-dried enzymepowder; the weight percent protein in the powder (20.3 wt %) wasdetermined using the BCA (Bicinchoninic acid) protein assay, and theglass transition temperature of this powder (Tg=54° C.) was measuredusing modulated differential scanning calorimetry. This solution wasspray-dried to produce a powder that was then tested for stabilityduring storage at 40° C. for 9 weeks. The spray-dried enzyme powder(stored at 40° C.) was sampled at one-week intervals and assayed foractivity using 50 μg protein/mL of T. neapolitana perhydrolase, H₂O₂(100 mM), triacetin (100 mM) and TURPINAL® SL (500 ppm) in 50 mMbicarbonate buffer (pH 7.2) at 25° C., and analyzed for production ofperacetic acid using a modification of the analytical method reported byKarst et al., supra. The perhydrolytic activity of the T. neapolitanaperhydrolase/maltodextrin spray-dried powder was stable over eight weeksof storage at 40° C. (Table 5).

TABLE 5 Temperature stability of T. neapolitanaperhydrolase/maltodextrin spray-dried enzyme powder during storage at40° C. PAA (ppm) produced in 5 min at 25° C. by reaction of triacetin(100 mM) and H₂O₂ (100 mM) in sodium bicarbonate buffer (50 mM, pH 7.2)containing T. neapolitana perhydrolase (50 μg protein/mL) and TURPINAL ®SL (500 ppm). time PAA (ppm) at 40° C. in 5 min initial 1142 week 1 1117week 2 1135 week 3 1087 week 4 964 week 5 1153 week 6 930 week 7 1025week 8 964

Example 11 Temperature Stability of T. neapolitanaPerhydrolase/Maltodextrin Spray-Dried Enzyme Powder Stored in a Mixtureof Enzyme Powder and Triacetin

The spray-dried enzyme powder prepared as described in Example 10 wasevaluated for stability when stored for twenty-one weeks at 40° C. as amixture of the spray-dried powder in triacetin. The spray-dried enzymepowder (1.235 g, 20.3 wt % protein) was added to 109 g of triacetin. Theresulting mixture was stored at 40° C., and a 2.19 g sample of thewell-stirred mixture assayed in duplicate at 25° C. in a 100-mL reactioncontaining hydrogen peroxide (100 mM) and TURPINAL® SL (500 ppm) in 50mM sodium bicarbonate buffer at pH 7.2, where the resultingconcentration of triacetin and protein was 100 mM and 50 μg/mL,respectively. Comparison of the data in Table 6 with the data in Example10, Table 5, demonstrates the stability of T. neapolitanaperhydrolase/maltodextrin spray-dried enzyme powders when stored as amixture with triacetin.

TABLE 6 Temperature stability of T. neapolitanaperhydrolase/maltodextrin spray-dried enzyme powder during storage in amixture of enzyme powder and triacetin at 40° C. PAA (ppm) produced in 5min at 25° C. by reaction of triacetin (100 mM) and H₂O₂ (100 mM) insodium bicarbonate buffer (50 mM, pH 7.2) containing T. neapolitanaperhydrolase (50 μg protein/mL) and TURPINAL ® SL (500 ppm). time PAA(ppm) in 5 min at 40° C. duplicate A duplicate B Average initial 10101019 1015 week 1 983 1054 1019 week 2 897 927 912 week 3 1194 1137 1166week 4 1139 1088 1114 week 5 1099 1069 1084 week 6 1098 978 1038 week 71018 1006 1012 week 8 907 892 900 week 12 925 936 931 week 18 824 NDweek 21 792 ND ND = a duplicate assay was not done

Example 12 Temperature Stability of T. maritimaperhydrolase/Maltodextrin Spray-Dried Enzyme Powder

An aqueous mixture was prepared containing heat-treated cell extractprotein of E. coli KLP18/pSW207 (ca. 21 g protein/L, ≧90% T. maritimaperhydrolase by PAGE) and maltodextrin (31 g/L maltodextrin DE 13-17 and31 g/L maltodextrin DE 4-7, Aldrich) as excipient in 50 mM sodiumbicarbonate (pH 8.1). This solution was spray-dried using a Buchi B-290glass-chamber spray dryer (inlet temperature=170° C., exittemperature=90° C., feed rate=4.5 mL/min) to produce a spray-driedenzyme powder; the weight percent protein in the powder (18.0 wt %) wasdetermined using the BCA (Bicinchoninic acid) protein assay, and theglass transition temperature of this powder (Tg=90° C.) was measuredusing modulated differential scanning calorimetry. This powder was thentested for stability during storage at 40° C. for 7 weeks. Thespray-dried enzyme powder (stored at 40° C.) was sampled at one-weekintervals and assayed for activity by adding 50 μg protein/mL of T.maritima perhydrolase to a reaction mixture containing H₂O₂ (100 mM),triacetin (100 mM) and TURPINAL® SL (500 ppm) in 50 mM bicarbonatebuffer (pH 7.2) at 25° C., and analyzed for production of peracetic acidusing a modification of the analytical method reported by Karst et al.The perhydrolytic activity of the T. maritima perhydrolase/maltodextrinspray-dried powder was stable over seven weeks of storage at 40° C.(Table 7).

TABLE 7 Temperature stability of T. maritima perhydrolase/maltodextrinspray- dried enzyme powder during storage at 40° C. PAA (ppm) producedin 5 min at 25° C. by reaction of triacetin (100 mM) and H₂O₂ (100 mM)in sodium bicarbonate buffer (50 mM, pH 7.2) containing T. maritimaperhydrolase (50 μg protein/mL) and TURPINAL ® SL (500 ppm). time PAA(ppm) at 40° C. in 5 min initial 1373 week 1 1262 week 2 1548 week 31317 week 4 1316 week 5 1378 week 6 1296 week 7 1475

Example 13 Temperature Stability of T. maritimaPerhydrolase/Maltodextrin Spray-Dried Enzyme Powder Stored in a Mixtureof Enzyme Powder and Triacetin

The spray-dried enzyme powder prepared as described in Example 12 wasevaluated for stability when stored for seven weeks at 40° C. as amixture of the spray-dried powder in triacetin. The spray-dried enzymepowder (0.556 g, 18.0 wt % protein) was added to 43.6 g of triacetin.The resulting mixture was stored at 40° C., and a 2.21 g sample of thewell-stirred mixture assayed in duplicate at 25° C. in a 100-mL reactioncontaining hydrogen peroxide (100 mM) and TURPINAL® SL (500 ppm) in 50mM sodium bicarbonate buffer at pH 7.2, where the resultingconcentrations of triacetin and protein were 100 mM and 50 μg/mL,respectively. Comparison of the data in Table 8 with the data in Example12, Table 7, demonstrates the stability of T. maritimaperhydrolase/maltodextrin spray-dried enzyme powders when stored as amixture with triacetin.

TABLE 8 Temperature stability of T. maritima perhydrolase/maltodextrinspray- dried enzyme powder during storage in a mixture of enzyme powderand triacetin at 40° C. PAA (ppm) produced in 5 min at 25° C. byreaction of triacetin (100 mM) and H₂O₂ (100 mM) in sodium bicarbonatebuffer (50 mM, pH 7.2) containing T. maritima perhydrolase (50 μgprotein/mL) and TURPINAL ® SL (500 ppm). time PAA (ppm) at 40° C. in 5min initial 1137 week 1 1089 week 2 1138 week 3 1213 week 4 1130 week 5872 week 6 858 week 7 1004

Example 14 Perhydrolysis of Propylene Glycol Diacetate or EthyleneGlycol Diacetate Using Bacillus subtilis ATCC® 31954™ Perhydrolase

A homogenate of a transformant expressing wild-type perhydrolase fromBacillus subtilis ATCC® 31954™ (KLP18/pSW194) was prepared from asuspension of cell paste (20 wt % wet cell weight) in 0.05 M potassiumphosphate buffer (pH 7.0) containing dithiothreitol (1 mM). The crudehomogenate was centrifuged to remove cellular debris, producing aclarified cell extract that was heat-treated at 65° C. for 30 min. Theresulting mixture was centrifuged, and the heat-treated supernatantconcentrated on a 30K MWCO (molecular weight cutoff) membrane to aconcentration of 32 mg/mL total dissolved solids; a SDS-PAGE of theclarified, heat-treated cell extract indicated that the perhydrolase wasat least 85-90% pure. To this concentrate was then added 2.06 grams ofNaH₂PO₄ and 1.17 grams Na₂HPO₄ per gram of solids was added to thisconcentrate to produce an approximate 3:1 ratio (wt/wt) of phosphatebuffer to heat-treated cell extract protein. This solution was dilutedby 30 wt % with deionized water, then spray-dried (180° C. inlettemperature, 70° C. exit temperature) using a Buchi B-290 laboratoryspray dryer); the resulting spray-dried powder contained 25.5 wt %protein (Bradford protein assay) and was 94.3 wt % dry solids.

Reactions (10 mL total volume) were run at 23° C. in 50 mM sodiumbicarbonate buffer (initial pH 7.2) containing propylene glycoldiacetate (PGDA) or ethylene glycol diacetate (EGDA), hydrogen peroxide(100 mM) and 123 μg/mL of a heat-treated extract protein from thespray-dried E. coli KLP18/pSW194 (expressing Bacillus subtilis ATCC®31954™ wild-type perhydrolase) (prepared as described above). A controlreaction for each reaction condition was run to determine theconcentration of peracetic acid produced by chemical perhydrolysis oftriacetin by hydrogen peroxide in the absence of added heat-treatedextract protein. The reactions were sampled at 1, 5, and 30 minutes andthe samples analyzed for peracetic acid using the Karst derivatizationprotocol (Karst et al., supra); aliquots (0.040 mL) of the reactionmixture were removed and mixed with 0.960 mL of 5 mM phosphoric acid inwater; adjustment of the pH of the diluted sample to less than pH 4immediately terminated the reaction. The resulting solution was filteredusing an ULTRAFREE® MC-filter unit (30,000 Normal Molecular Weight Limit(NMWL), Millipore cat #UFC3LKT 00) by centrifugation for 2 min at 12,000rpm. An aliquot (0.100 mL) of the resulting filtrate was transferred to1.5-mL screw cap HPLC vial (Agilent Technologies, Palo Alto, Calif.;#5182-0715) containing 0.300 mL of deionized water, then 0.100 mL of 20mM MTS (methyl-p-tolyl-sulfide) in acetonitrile was added, the vialscapped, and the contents briefly mixed prior to a 10 min incubation atca. 25° C. in the absence of light. To each vial was then added 0.400 mLof acetonitrile and 0.100 mL of a solution of triphenylphosphine (TPP,40 mM) in acetonitrile, the vials re-capped, and the resulting solutionmixed and incubated at ca. 25° C. for 30 min in the absence of light. Toeach vial was then added 0.100 mL of 10 mM N,N-diethyl-m-toluamide(DEET; HPLC external standard) and the resulting solution analyzed byHPLC. The peracetic acid concentrations produced in 1 min, 5 min and 30min are listed in Table 9.

TABLE 9 Peracetic acid (PAA) concentration produced in reactionsutilizing propylene glycol diacetate (PGDA) or ethylene glycol diacetate(EGDA) and hydrogen peroxide (100 mM) in sodium bicarbonate buffer (50mM, initial pH 7.2) at 23° C. using 123 μg/mL of heat-treated extractprotein from E. coli KLP18/pSW194 (Bacillus subtilis ATCC ® 31954 ™perhydrolase). PAA, PAA, PAA, perhydrolase substrate 1 min 5 min 30 min(50 μg/mL) (100 mM) (ppm) (ppm) (ppm) no enzyme (control) PGDA 0 64 241B. subtilis ATCC ® 31954 PGDA 666 781 815 no enzyme (control) EGDA 0 18141 B. subtilis ATCC ® 31954 EGDA 747 931 963

Example 15 Perhydrolysis of Propylene Glycol Diacetate or EthyleneGlycol Diacetate Using T. maritima and T. neapolitana Wild-type andVariant Perhydrolases

Cell extracts of transformants expressing Thermotoga neapolitanawild-type perhydrolase (KLP18/pSW196), Thermotoga neapolitana C277Svariant perhydrolase (KLP18/pSW196/C277S), Thermotoga neapolitana C277Tvariant perhydrolase (KLP18/pSW196/C277T), Thermotoga maritima wild-typeperhydrolase (KLP18/pSW228), Thermotoga maritima C277S variantperhydrolase (KLP18/pSW228/C277S), and Thermotoga maritima C277T variantperhydrolase (KLP18/pSW228/C277T) were each prepared by passing asuspension of cell paste (20 wt % wet cell weight) in 0.05 M potassiumphosphate buffer (pH 7.0) containing dithiothreitol (1 mM) twice througha French press having a working pressure of 16,000 psi (˜110 MPa). Thelysed cells were centrifuged for 30 minutes at 12,000×g, producing aclarified cell extract that was assayed for total soluble protein(Bradford assay). The supernatant was heated at 75° C. for 20 minutes,followed by quenching in an ice bath for 2 minutes. Precipitated proteinwas removed by centrifugation for 10 minutes at 11,000×g. SDS-PAGE ofthe resulting heat-treated extract protein supernatant indicated thatthe CE-7 enzyme comprised approximately 85-90% of the total protein inthe preparation. The heat-treated extract protein supernatant was frozenin dry ice and stored at −80° C. until use.

A first set of reactions (10 mL total volume) were run at 20° C. in 10mM sodium bicarbonate buffer (initial pH 8.1) containing propyleneglycol diacetate (PGDA) or ethylene glycol diacetate (EGDA) (100 mM),hydrogen peroxide (100 mM) and 25 μg/mL of heat-treated extract proteinfrom one of E. coli KLP18/pSW196 (Thermotoga neapolitana wild-typeperhydrolase), E. coli KLP18/pSW196/C277S (Thermotoga neapolitana C277Svariant perhydrolase), E. coli KLP18/pSW196/C277T (Thermotoganeapolitana C277T variant perhydrolase), E. coli KLP18/pSW228(Thermotoga maritima wild-type perhydrolase), E. coli KLP18/pSW228/C277S(Thermotoga maritima C277S variant perhydrolase), and E. coliKLP18/pSW228/C277T (Thermotoga maritima C277T variant perhydrolase)(prepared as described above). A control reaction for each reactioncondition was run to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. The reactions were sampled at 1,5, and 30 minutes and the samples analyzed for peracetic acid using theKarst derivatization protocol (Karst et al., supra) and HPLC analyticalmethod (supra). The peracetic acid concentrations produced in 1 min, 5min and 30 min are listed in Table 10.

TABLE 10 Peracetic acid (PAA) concentration produced utilizing T.maritima and T. neapolitana wild-type and variant perhydrolases inreactions at 20° C. in sodium bicarbonate buffer (10 mM, initial pH 8.1)containing propylene glycol diacetate (PGDA) (100 mM) or ethylene glycoldiacetate (EGDA) (100 mM), hydrogen peroxide (100 mM) and 25 μg/mL ofheat-treated extract protein. sub- strate PAA, PAA, PAA, sub- conc. H₂O₂1 min 5 min 30 min perhydrolase strate (mM) (mM) (ppm) (ppm) (ppm) noenzyme (control) PGDA 100 100 0 15 165 T. maritima WT PGDA 100 100 5341104 1695 T. maritima C277S PGDA 100 100 647 1320 1864 T. maritima C277TPGDA 100 100 656 1174 1418 T. neapolitana WT PGDA 100 100 513 1052 1946T. neapolitana PGDA 100 100 875 1327 1707 C277S T. neapolitana PGDA 100100 724 1325 1864 C277T no enzyme (control) EGDA 100 100 0 70 229 T.maritima WT EGDA 100 100 765 1182 1595 T. maritima C277S EGDA 100 100725 1240 1724 T. maritima C277T EGDA 100 100 802 1218 1734 T.neapolitana WT EGDA 100 100 603 1132 1643 T. neapolitana EGDA 100 100680 1305 1698 C277S T. neapolitana EGDA 100 100 688 1164 1261 C277T

A second set of reactions (10 mL total volume) were run at 20° C. in 10mM sodium bicarbonate buffer (initial pH 8.1) containing propyleneglycol diacetate (PGDA) or ethylene glycol diacetate (EGDA) (2 mM),hydrogen peroxide (10 mM) and 10 μg/mL of heat-treated extract proteinfrom one of E. coli KLP18/pSW196 (Thermotoga neapolitana wild-typeperhydrolase), E. coli KLP18/pSW196/C277S (Thermotoga neapolitana C277Svariant perhydrolase), E. coli KLP18/pSW196/C277T (Thermotoganeapolitana C277T variant perhydrolase), E. coli KLP18/pSW228(Thermotoga maritima wild-type perhydrolase), E. coli KLP18/pSW228/C277S(Thermotoga maritima C277S variant perhydrolase), and E. coliKLP18/pSW228/C277T (Thermotoga maritima C277T variant perhydrolase)(prepared as described above). A control reaction for each reactioncondition was run to determine the concentration of peracetic acidproduced by chemical perhydrolysis of triacetin by hydrogen peroxide inthe absence of added extract protein. The reactions were sampled at 5minutes and the samples analyzed for peracetic acid using the Karstderivatization protocol (Karst et al., supra) and HPLC analytical method(supra). The peracetic acid concentrations produced in 5 min are listedin Table 11.

TABLE 11 Peracetic acid (PAA) concentration produced utilizing T.maritima and T. neapolitana wild-type and variant perhydrolases inreactions at 20° C. in sodium bicarbonate buffer (10 mM, initial pH 8.1)containing propylene glycol diacetate (PGDA) (2 mM) or ethylene glycoldiacetate (EGDA) (2 mM), hydrogen peroxide (10 mM) and 10 μg/mL ofheat-treated extract protein. substrate PAA, conc. H₂O₂ 5 minperhydrolase substrate (mM) (mM) (ppm) no enzyme (control) PGDA 2 10 3.6T. maritima WT PGDA 2 10 5.0 T. maritima C277S PGDA 2 10 7.2 T. maritimaC277T PGDA 2 10 7.9 T. neapolitana WT PGDA 2 10 5.7 T. neapolitana C277SPGDA 2 10 7.9 T. neapolitana C277T PGDA 2 10 3.9 no enzyme (control)EGDA 2 10 3.3 T. maritima WT EGDA 2 10 9.9 T. maritima C277S EGDA 2 1013.6 T. maritima C277T EGDA 2 10 22.9 T. neapolitana WT EGDA 2 10 6.6 T.neapolitana C277S EGDA 2 10 18.4 T. neapolitana C277T EGDA 2 10 20.2

1. A process to stabilize the perhydrolysis activity of an enzyme whenpresent in a formulation comprised of said enzyme and a carboxylic acidester, the process comprising: (a) providing an aqueous formulationcomprising: (i) at least one enzyme structurally classified as a CE-7enzyme and having perhydrolysis activity and a signature motifcomprising: (1) an RGQ motif at amino acid residues 118-120, (2) a GXSQGmotif at residues 179-183, and (3) an HE motif at residues 298-299 whenaligned to reference sequence SEQ ID NO:1 using CLUSTALW, said enzymefurther having at least 30% amino acid identity to SEQ ID NO:1, (ii) atleast one oligosaccharide excipient, and (iii) optionally at least onesurfactant; and (b) spray-drying the aqueous formulation of (a) toproduce an enzyme powder which substantially retains the perhydrolysisactivity of the at least one enzyme when present in a formulationcomprised of a carboxylic acid ester and the enzyme powder.
 2. Theprocess of claim 1, wherein the at least one oligosaccharide excipienthas a number average molecular weight of at least about 1250 and aweight average molecular weight of at least about
 9000. 3. The processof claim 2, wherein the at least one oligosaccharide excipient isselected from the group consisting of maltodextrin, xylan, mannan,fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin,levan, graminan, amylopectin, and mixtures thereof.
 4. The process ofclaim 3, wherein the at least one oligosaccharide excipient ismaltodextrin.
 5. The process of claim 1, wherein the at least oneoligosaccharide excipient is trehalose.
 6. The process of claim 1,wherein the carboxylic acid ester is selected from the group consistingof monoacetin, diacetin, triacetin, monopropionin, dipropionin,tripropionin, monobutyrin, dibutyrin, tributyrin, and mixtures thereof.7. The process of claim 6, wherein the carboxylic acid ester istriacetin.
 8. The process of claim 1, wherein the at least onesurfactant is present and is polysorbate
 80. 9. The process of claim 2,wherein the at least one oligosaccharide excipient has a number averagemolecular weight of at least about 1700 and a weight average molecularweight of at least about
 15000. 10. The process of claim 1, wherein theat least one enzyme comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, and SEQ IDNO:20, wherein amino acid residue 277 of SEQ ID NO: 19 or SEQ ID NO: 20is selected from the group consisting of alanine, valine, serine, andthreonine. 11-21. (canceled)